CN110894492A - Pancreatic cancer in-vitro 3D model construction method based on pancreatic acellular scaffold - Google Patents
Pancreatic cancer in-vitro 3D model construction method based on pancreatic acellular scaffold Download PDFInfo
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Abstract
A method for constructing a pancreatic cancer in-vitro 3D model based on a pancreatic acellular scaffold belongs to the technical field of tissue engineering and tumor invasion, metastasis and drug resistance, and comprises the steps of simulating a tumor in-vivo microenvironment based on the pancreatic acellular scaffold, replanting pancreatic cancer cells, constructing a pancreatic cancer in-vitro 3D culture model, introducing a cell-extracellular matrix and a three-dimensional space structure on the basis of cell-cell interaction in the traditional 2D culture, enabling tumor cells to grow in the three-dimensional space structure, showing circular and oval cell shapes, and showing changes of a nuclear-cytoplasmic ratio and a nuclear fission image.
Description
Technical Field
The invention belongs to the technical field of tissue engineering, tumor invasion, metastasis and drug resistance, and particularly relates to a method for constructing a pancreatic cancer in-vitro 3D model based on a pancreatic acellular scaffold.
Background
Pancreatic cancer is a malignant tumor seriously harming human health, the incidence rate of the pancreatic cancer is increased in recent years, the malignancy degree of the pancreatic cancer is high, and the five-year survival rate of pancreatic cancer patients is not more than 9% according to recent reports. Most patients are found to be in an advanced stage, the complete tumor resection rate is low, the overall prognosis is poor, and therefore, the research on pancreatic cancer still has great clinical value. In the past, studies on tumor cell characteristics, invasiveness, drug resistance, etc. have generally been based on in vitro 2D culture of tumor cells and tumor xenograft models, however, 2D tumor cell culture models have failed to provide an appropriate tumor microenvironment due to a lack of appropriate three-dimensional (3D) structures of cell-cell and cell-extracellular matrix interactions. Tumor xenograft models can mimic the human tumor microenvironment, however, animal experiments involve many uncontrollable factors including hemodynamics, host cells, endogenous growth factors, and immune responses. Furthermore, monitoring the therapeutic response of tumor xenograft models is expensive and difficult. In recent years, through a gene modification technical method, a mode of directly replacing a mouse-related gene with a human-related gene to establish a humanized mouse model has been widely applied to the research in the biomedical fields such as human gene function research, tumor immune drug development, infectious disease establishment, preclinical evaluation of drugs and the like, but the method has the defects of complex operation, high price, high experimental condition requirements and the like, and still cannot be widely applied to the basic research of pancreatic cancer.
Studies in recent years have clearly demonstrated that cells behave differently in two-or three-dimensional media, and efforts have therefore been made to develop new tumor models that reproduce the natural microenvironment of the tumor as accurately as possible. The 3D in vitro culture model is used to replicate tumor-specific cells and tumor microenvironments and provides an important choice for 2D monolayer culture and complex in vivo xenograft methods. The three-dimensional culture model provides a controllable tumor microenvironment simulating the in vivo growth, invasion and metastasis of tumor cells, and growth factors, extracellular matrix proteins, cell types and the like are artificially controlled in the environment.
In the past decade, tissue decellularization (the process of removing cells without affecting the extracellular matrix structure and composition) has emerged as an alternative technology in the biomedical field, tissue engineering and regenerative medicine. Organs such as heart, lung, liver and the like are successfully decellularized and are transplanted again by cells, so that the problem of end organ failure is solved. In recent years, tissue decellularized scaffolds have also been used to elucidate the complex role of extracellular matrix in tumor growth, metastasis and progression. The acellular scaffold reserves the biomechanical characteristics of natural tissues and unique extracellular matrix composition and structure, and can be used as a platform of tumor engineering.
The invention adopts normal pancreatic tissues of rats, the tissues are effectively decellularized and are refilled with human pancreatic cancer cells, a tumor three-dimensional scaffold with ideal spatial arrangement, biomechanical property and biocompatibility is constructed, and an in-vitro model simulating a pancreatic cancer microenvironment is established. The description and related experiments prove that the pancreatic acellular scaffold can be used as an ideal in-vitro pancreatic cancer engineering scaffold, and the model aims to clarify the effect of the pancreatic acellular scaffold on pancreatic cancer cells, the influence of the pancreatic acellular scaffold on the proliferation, invasion, metastasis, drug resistance and other biological characteristics of the pancreatic cancer cells and the research on related mechanisms, and is further used for screening pancreatic cancer chemotherapeutic drugs and formulating individualized clinical chemotherapeutic schemes.
Disclosure of Invention
The invention mainly solves the technical problems in the prior art and provides a method for constructing a pancreatic cancer in-vitro 3D model based on a pancreatic acellular scaffold.
The technical problem of the invention is mainly solved by the following technical scheme: a method for constructing an in-vitro 3D model of pancreatic cancer based on a pancreatic acellular scaffold, wherein the pancreatic acellular scaffold is derived from mouse, rat and pig pancreas, and the pancreatic acellular scaffold is used as an in-vitro tumor model, and the method comprises the following steps:
step 1, sterilizing a pancreas acellular scaffold by cobalt-60 irradiation, and incubating for 5 minutes in a culture medium at 37 ℃ after sterilization;
step 2, 1 × 107The pancreatic cancer cell number is digested by pancreatin, added with 4ml of culture medium, mixed well, slowly injected through the spleen artery for 4 times, 1ml each time, at 15 min intervals, and treated with 5% CO at 37 deg.C2And standing in the incubator for 2 hours, performing 1ml/min circulation perfusion, replacing the culture medium every day, and performing circulation perfusion culture for 5 days to obtain the pancreatic cancer in-vitro 3D model based on the pancreatic acellular scaffold.
Preferably, the pancreatic cancer cell lines in step 2 include HPAC, HuP-T4, BxPC-3, Capan-1, Miapaca-2, PANC-1, HS 766T, CFPAC-1, HuP-T3, HPAF-II, KP-4, Panc02.03, Panc 03.27, Panc 04.03, Panc08.13, QGP-1, YAPC, SU.86.86, PK-59, PSN1, SW1990, T3M-4, Capan-2, Panc 10.05.
Preferably, the culture medium of the HPAC is DMEM +0.002mg/ml insulin +0.005mg/ml transferrin +40ng/ml hydrocortisone +10ng/ml epidermal growth factor + 5% FBS, the culture medium of the HuP-T4 is MEM + 20% FBS + 1% NEAA + 1% NaP, the culture medium of the BxPC-3 and Capan-1 is IMDM + 20% FBS, the culture medium of the Miapaca-2 is DMEM + 10% FBS + 2.5% horse serum, the culture medium of the PANC-1 and HS 766T is: DMEM + 10% FBS, the culture media of CFPAC-1, HuP-T3 and HPAF-II are MEM + 10% FBS + 1% NEAA + 1% NaP, and the culture media of KP-4, Panc02.03, Panc 03.27, Panc 04.03, Panc08.13, QGP-1, YAPC, SU.86.86, PK-59 and PSN1 are: RPMI-1640+ 10% FBS, the medium of SW1990 is L-15+ 10% FBS, the medium of T3M-4 is HamF10+ 10% FBS, the medium of Capan-2 is McCoy's 5a + 10% FBS, and the medium of Panc 10.05 is RPMI-1640+10Units/ml human + 15% FBS.
A method for preparing pancreatic cancer cell suspension by using an in-vitro pancreatic cancer 3D model, which comprises the following steps:
step 1, carrying out pancreatic cancer cell colonization, observation of cell morphology and proliferation detection on a pancreatic cancer in-vitro 3D model, wherein the pancreatic cancer in-vitro 3D model is subjected to paraffin embedding, continuous slicing, section dewaxing, HE and Ki67 immunohistochemical staining and mounting observation;
and 2, obtaining pancreatic cancer cells, extracting by trypsinization, cutting the pancreatic acellular scaffold planted with the pancreatic cancer cells into small blocks, digesting by 1% trypsin at 37 ℃ for 30 minutes, shaking mutually, adding a culture medium containing 10% FBS to terminate the reaction, centrifuging the solution at 300g for 10 minutes, filtering the solution containing the cells through a BD-Falcon cell filter with the pore diameter of 70 mu m to obtain single separated cells, and re-suspending the obtained cells to obtain a 3D cultured pancreatic cancer cell suspension.
Preferably, the pancreatic cancer cell suspension is applied to cell functional experiments, drug resistance detection and related mechanism research.
The invention has the following beneficial effects:
the invention discloses a pancreatic cancer in-vitro 3D culture model constructed by simulating a tumor in-vivo microenvironment based on a pancreatic decellularized scaffold and replanting pancreatic cancer cells, wherein the pancreatic cancer in-vitro 3D culture model is constructed by introducing a cell-extracellular matrix and a three-dimensional space structure on the basis of cell-cell interaction in the traditional 2D culture, tumor cells can grow in the three-dimensional space structure, show circular and oval cell forms and can see the change of nuclear mass ratio and nuclear fission.
Drawings
FIG. 1 is a graph of HE staining of normal rat pancreatic tissue with intact pancreatic leaflet structure and normal cells;
FIG. 2 is a graph of DAPI staining after pancreatic decellularized scaffold sections from normal rats;
FIG. 3 is a graph of HE staining after sectioning of a pancreatic decellularized scaffold of the invention;
FIG. 4 is a staining pattern of a sectioned decellularized pancreatic scaffold of the invention;
FIG. 5 is a graph of HE staining of sections after replanting of Panc-1 cells of the invention;
FIG. 6 is an under 400X magnification of a slice HE staining after replanting Panc-1 cells of the invention;
FIG. 7 is a Ki67 immunohistochemistry plot following scaffold sectioning of Panc-1 cells of the invention;
FIG. 8 is a graph showing the migration experiment of Panc-1 cells cultured in 3D according to the present invention;
FIG. 9 is a graph showing a conventional 2D-cultured Panc-1 cell migration experiment;
FIG. 10 is a graph comparing the number of cells migrated in one experiment of Panc-1 cells cultured in 3D according to the present invention with that of Panc-1 cells cultured in 2D according to the prior art;
FIG. 11 is a graph showing the invasion assay of Panc-1 cells cultured in 3D according to the present invention;
FIG. 12 is a graph showing the experiment of Panc-1 cell invasion in conventional 2D culture;
FIG. 13 is a graph showing the comparison of the number of cell invasion in one experiment of Panc-1 cells cultured in 3D and Panc-1 cells cultured in 2D according to the present invention.
Detailed Description
The technical scheme of the invention is further specifically described by the following embodiments and the accompanying drawings.
Example (b): a method for constructing an in vitro 3D model of pancreatic cancer based on a pancreatic decellularized scaffold derived from mouse, rat and pig pancreas as an in vitro tumor model, as shown in fig. 1-13, the method comprising:
step 1, sterilizing a pancreas acellular scaffold by cobalt-60 irradiation, and incubating for 5 minutes in a culture medium at 37 ℃ after sterilization;
step 2, 1 × 107The pancreatic cancer cell number is digested by pancreatin, 4ml of culture medium is added, the mixture is fully and uniformly mixed, slow injection is carried out for 4 times through a spleen artery, 1ml of the injection is carried out each time at intervals of 15 minutes, the mixture is stood in a 5% CO2 incubator at 37 ℃ for 2 hours and then is subjected to 1ml/min circulation perfusion, the culture medium is changed every day, and circulation perfusion culture is carried out for 5 days, so that the pancreatic cancer in-vitro 3D model based on the pancreatic acellular scaffold is obtained.
The pancreatic cancer cell lines in step 2 include HPAC, HuP-T4, BxPC-3, Capan-1, Miapaca-2, PANC-1, HS 766T, CFPAC-1, HuP-T3, HPAF-II, KP-4, Panc02.03, Panc 03.27, Panc 04.03, Panc08.13, QGP-1, YAPC, SU.86.86, PK-59, PSN1, SW1990, T3M-4, Capan-2, Panc 10.05.
The culture medium of HPAC is DMEM +0.002mg/ml insulin +0.005mg/ml transferrin +40ng/ml hydrocortisone +10ng/ml epidermal growth factor + 5% FBS, the culture medium of HuP-T4 is MEM + 20% FBS + 1% NEAA + 1% NaP, the culture medium of BxPC-3 and Capan-1 is IMDM + 20% FBS, the culture medium of Miapaca-2 is DMEM + 10% FBS + 2.5% horse serum, the culture medium of PANC-1 and HS 766T is: DMEM + 10% FBS, the culture media of CFPAC-1, HuP-T3 and HPAF-II are MEM + 10% FBS + 1% NEAA + 1% NaP, and the culture media of KP-4, Panc02.03, Panc 03.27, Panc 04.03, Panc08.13, QGP-1, YAPC, SU.86.86, PK-59 and PSN1 are: RPMI-1640+ 10% FBS, the medium of SW1990 is L-15+ 10% FBS, the medium of T3M-4 is HamF10+ 10% FBS, the medium of Capan-2 is McCoy's 5a + 10% FBS, and the medium of Panc 10.05 is RPMI-1640+10Units/ml human + 15% FBS.
A method for preparing pancreatic cancer cell suspension by using an in-vitro pancreatic cancer 3D model, which comprises the following steps:
step 1, carrying out pancreatic cancer cell colonization, observation of cell morphology and proliferation detection on a pancreatic cancer in-vitro 3D model, wherein the pancreatic cancer in-vitro 3D model is subjected to paraffin embedding, continuous slicing, section dewaxing, HE and Ki67 immunohistochemical staining and mounting observation;
and 2, obtaining pancreatic cancer cells, extracting by trypsinization, cutting the pancreatic acellular scaffold planted with the pancreatic cancer cells into small blocks, digesting by 1% trypsin at 37 ℃ for 30 minutes, shaking mutually, adding a culture medium containing 10% FBS to terminate the reaction, centrifuging the solution at 300g for 10 minutes, filtering the solution containing the cells through a BD-Falcon cell filter with the pore diameter of 70 mu m to obtain single separated cells, and re-suspending the obtained cells to obtain a 3D cultured pancreatic cancer cell suspension.
The pancreatic cancer cell suspension is applied to cell functional experiments, drug resistance detection and related mechanism research.
The present invention will be further described with reference to the following specific examples.
1. Materials and reagents
A pancreatic decellularized scaffold (prepared using the method described in the applicant's issued patent No. ZL201410590216.8 invention) derived from rat or porcine pancreas, in this example using rat pancreas and Panc-1 cells; trypsin, fetal bovine serum (Gibco).
The main apparatus comprises: three-dimensional dynamic circulation perfusion device (Equl), peristaltic pump, cell culture case (Thermo) and biological safety cabinet.
2. The procedure for preparing the 3D tumor model was performed:
1) pancreatic decellularized scaffolds were sterilized by cobalt-60 irradiation and incubated in 37 ℃ medium for 5 minutes.
2) Will be 1 × 107Pancreatic cancer cell number, adding 4ml culture medium after pancreatin digestion, mixing well, slowly injecting 4 times through spleen artery, 1ml each time, 15 min apart, 5% CO at 37 deg.C2Standing in an incubator for 2 hours, performing 1ml/min circulation perfusion, replacing culture medium every day, and performing circulation perfusion culture for 5 days to obtain pancreatic cancer based on the pancreatic acellular scaffoldAn outer 3D model.
3) Pancreatic cancer cell colonization, cell morphology observation and proliferation detection on the 3D model: the model was paraffin embedded, serial sectioned, section deparaffinized, HE and Ki67 immunohistochemical stained, mounted for viewing.
4) The pancreatic cancer cell acquisition is as follows: the pancreatic cancer cell-free scaffold planted with pancreatic cancer cells is cut into small pieces by trypsinization extraction, digested with 1% trypsin at 37 ℃ for 30 minutes, shaken with each other, added with a culture medium containing 10% FBS to terminate the reaction, centrifuged at 300g for 10 minutes, the solution containing the cells is filtered through a BD-Falcon cell filter with a pore size of 70 μm to obtain single isolated cells, and the obtained cells are resuspended to obtain a pancreatic cancer cell suspension, namely a 3D-cultured pancreatic cancer cell suspension.
5) The obtained pancreatic cancer cells cultured by 3D are used for carrying out corresponding cell functional experiments, drug resistance detection and related mechanism research, and migration experiments and invasion experiments are taken as examples. 3D and 2D cultured pancreatic cancer cells were cultured overnight in DMEM without FBS, 3X 104A cell suspension of 100. mu.L (not containing FBS) at/ML density was placed in the upper chamber layer, and 750. mu.L MEM containing 20% FBS was placed in the lower chamber layer. 5% CO at 37 deg.C2After 12 hours of incubation in the incubator, formaldehyde was fixed for 20 minutes and the cells on the inner surface of the chamber were cleaned with a cotton swab. The cells were stained with 0.1% crystal violet for 10 minutes and observed by an optical microscope to count the number of migrated cells. The invasion assay procedure was essentially the same as the migration assay except that the chamber was pre-coated with matrigel.
It can be seen in fig. 1 and 2 that the cells within the pancreatic tissue are completely removed; FIGS. 3 and 4 show that the decellularized scaffold retains extracellular matrix components; FIGS. 5 and 6 show that Panc-1 cells colonize on the decellularized scaffold, and the morphology of the cells changes, and the karyoplasmic ratio and the nuclear fission image also change; the proliferation of Panc-1 cells on pancreatic decellularized scaffolds is demonstrated in fig. 7; it can be seen in FIGS. 8, 9 and 10 that the migration ability of Panc-1 cells cultured in 3D is strong; FIGS. 11, 12 and 13 show that Panc-1 cells cultured in 3D have a greater invasive potential.
Finally, it should be noted that the above embodiments are merely representative examples of the present invention. It will be clear that the invention is not limited to the embodiments described above, but that many variations and more extensive and intensive investigations are possible. Any simple modification, equivalent change and modification made to the above embodiments in accordance with the technical spirit of the present invention should be considered to be within the scope of the present invention.
Claims (5)
1. A method for constructing an in vitro 3D model of pancreatic cancer based on a pancreatic acellular scaffold, wherein the pancreatic acellular scaffold is derived from mouse, rat and pig pancreas, and the pancreatic acellular scaffold is used as an in vitro tumor model, and is characterized in that the method for constructing the in vitro 3D model of pancreatic cancer comprises the following steps:
step 1, sterilizing a pancreas acellular scaffold by cobalt-60 irradiation, and incubating for 5 minutes in a culture medium at 37 ℃ after sterilization;
step 2, 1 × 107The pancreatic cancer cell number is digested by pancreatin, added with 4ml of culture medium, mixed well, slowly injected through the spleen artery for 4 times, 1ml each time, at 15 min intervals, and treated with 5% CO at 37 deg.C2And standing in the incubator for 2 hours, performing 1ml/min circulation perfusion, replacing the culture medium every day, and performing circulation perfusion culture for 5 days to obtain the pancreatic cancer in-vitro 3D model based on the pancreatic acellular scaffold.
2. The method for constructing the pancreatic cancer in-vitro 3D model based on the pancreatic acellular stent of claim 1, wherein the pancreatic cancer cell lines in the step 2 comprise HPAC, HuP-T4, BxPC-3, Capan-1, Miapaca-2, PANC-1, HS 766T, CFPAC-1, HuP-T3, HPAF-II, KP-4, Panc02.03, Panc 03.27, Panc 04.03, Panc08.13, QGP-1, YAPC, SU.86.86, PK-59, PSN1, SW1990, T3M-4, Capan-2 and Panc 10.05.
3. The method for constructing an in vitro 3D model of pancreatic cancer based on pancreatic decellularized scaffold, according to claim 2, wherein the culture medium of HPAC is DMEM +0.002mg/ml insulin +0.005mg/ml transferrin +40ng/ml hydrocortisone +10ng/ml epidermal growth factor + 5% FBS, the culture medium of HuP-T4 is MEM + 20% FBS + 1% NEAA + 1% NaP, the culture medium of BxPC-3 and Capan-1 is IMDM + 20% FBS, the culture medium of Miapaca-2 is DMEM + 10% FBS + 2.5% horse um, the culture medium of PANC-1 and HS 766T is: DMEM + 10% FBS, the culture media of CFPAC-1, HuP-T3 and HPAF-II are MEM + 10% FBS + 1% NEAA + 1% NaP, and the culture media of KP-4, Panc02.03, Panc 03.27, Panc 04.03, Panc08.13, QGP-1, YAPC, SU.86.86, PK-59 and PSN1 are: RPMI-1640+ 10% FBS, the medium of SW1990 is L-15+ 10% FBS, the medium of T3M-4 is HamF10+ 10% FBS, the medium of Capan-2 is McCoy's 5a + 10% FBS, and the medium of Panc 10.05 is RPMI-1640+10Units/ml human + 15% FBS.
4. A method for preparing pancreatic cancer cell suspension by using an in-vitro pancreatic cancer 3D model is characterized by comprising the following steps:
step 1, carrying out pancreatic cancer cell colonization, observation of cell morphology and proliferation detection on a pancreatic cancer in-vitro 3D model, wherein the pancreatic cancer in-vitro 3D model is subjected to paraffin embedding, continuous slicing, section dewaxing, HE and Ki67 immunohistochemical staining and mounting observation;
and 2, obtaining pancreatic cancer cells, extracting by trypsinization, cutting the pancreatic acellular scaffold planted with the pancreatic cancer cells into small blocks, digesting by 1% trypsin at 37 ℃ for 30 minutes, shaking mutually, adding a culture medium containing 10% FBS to terminate the reaction, centrifuging the solution at 300g for 10 minutes, filtering the solution containing the cells through a BD-Falcon cell filter with the pore diameter of 70 mu m to obtain single separated cells, and re-suspending the obtained cells to obtain a 3D cultured pancreatic cancer cell suspension.
5. The method for preparing pancreatic cancer cell suspension using in vitro 3D model of pancreatic cancer according to claim 4, wherein the pancreatic cancer cell suspension is applied to cytofunctional experiments, drug resistance detection and related mechanism research.
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