CN108795867A - The method for shifting external threedimensional model for building colon cancer cell peritonaeum - Google Patents
The method for shifting external threedimensional model for building colon cancer cell peritonaeum Download PDFInfo
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Abstract
The method that the present invention relates to a kind of to shift external threedimensional model for building colon cancer cell peritonaeum, including step：(1) fibroblast suspension is mixed with type i collagen, is added in culture vessel immediately after mixing；(2) culture vessel is placed at room temperature 20 minutes, is subsequently placed in cell incubator and is solidified after 2~4 hours, continue to spread mesothelial cell in plate 18~38 hours；(3) colon cancer cell is spread in plate, the structure of the outer 3D models of perfect aspect.Use the present invention for building the method that colon cancer cell peritonaeum shifts external threedimensional model, physiology microenvironment can be simulated, variation for more fully understanding adherency and invasive ability in colorectal cancer transfer process, the current in-vitro cell culture model in research colorectal cancer metastasis of cancer will be improved, and helps to enhance the overall understanding to colorectal cancer early stage transfer process.
Description
Technical field
It is external three-dimensional for building the transfer of colon cancer cell peritonaeum that the present invention relates to biotechnologies, in particular to one kind
The method of model.
Background technology
More than 20 years, the colon cancer morbidity of China and other economic transformation type countries was constantly rising, main reasons is that
Caused by the life of environmental factor and people and the change of eating habit.Colorectal cancer have become world's common cancer it
One, incidence accounts for the 3rd colon cancer of gastroenteric tumor, and recurrence and transfer are clinically urgent problems.
It is directly planted caused by growth through the transfer of bloody path peritonaeum or peritonaeum the study found that peritonaeum transfer is cancer cell.
In the case that clinic shows that peritonaeum transfer occurs in patient, progression of the disease quickening, poor prognosis need to use combination therapy measure more.Stomach
Why intestinal canal tumour cell is easy to happen operation posterior peritoneum transfer, is to be covered with an interlayer chrotoplast because of cavum peritoneale surface,
When the tumour cell to fall off touches mesothelial cell, it will be adhered to above, invade peritonaeum, gradually form new tumour.Though
The animal model so having built up can be used for the screening of internal drug, but in vitro study and getting up early molecular marker
Discovery there are still certain drawbacks.It is an extremely complex process in view of metastases, it includes extracellular matrix, cell
Several aspects such as adhesion property change, Tumor angiogenesis, cell survival.
The culture of two-dimentional (2D) superficial cell provides breakthrough opinion for basic cell biology and tumour.So
And under conditions of these simplification, most of physiological parameters of organ or tumour (such as institutional framework, cell is with cell and carefully
The interaction of born of the same parents and matrix, engineering properties and biochemistry network) all lose.
All in the interaction between detecting cancer cell, extracellular matrix and mesothelial cell are also required into one recent study
In the addition research of step.Extracellular matrix or the in vitro culture experiment of mesothelial cell are completed currently without research.Also, current
There is no effect of the clear fibroblast in cancer cell adhesion and invasive procedure for research.In addition, most of researchs only carry out
Then research of the cancer metastasis to peritonaeum only speculates that the mechanism of action of transfer is to be with other transfers (e.g., hepatic metastases)
It is similar.However, not knowing why colon cancer is there are one the tendency to peritonaeum transfer, and why colon cancer is transferred at present
Peritonaeum is easier to occur than other sites.
This invention address that colon cancer strokes the external threedimensional model of transfer in one novel analogue body of proposition, for grinding
Study carefully the existing effect of microenvironment and the searching and exploration of early stage colon cancer Metastatic Marker in colon cancer peritonaeum transfer process.
Invention content
It is improved in view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide one kind for studying colorectal cancer metastasis of cancer
In-vitro cell culture model and contributing to enhance to the overall understanding of colorectal cancer early stage transfer process for building knot
The method that colon-cancer cell peritonaeum shifts external threedimensional model.
To achieve the goals above, of the invention for building the method that colon cancer cell peritonaeum shifts external threedimensional model
It is as follows：
The method includes step：
(1), fibroblast suspension is mixed with type i collagen, is added in culture vessel immediately after mixing；
(2), culture vessel is placed at room temperature 20 minutes, is subsequently placed in cell incubator after 2~4 hours and coagulates
Gu continuing to spread mesothelial cell in plate 18~38 hours；
(3) colon cancer cell is spread in plate, the structure of the outer 3D models of perfect aspect.
Preferably, the fibroblast in the step (1) is primary peritoneal fibroblasts；The step (1)
In type i collagen be I type rat tail collagen proteins；Mesothelial cell in the step (2) is primary Peritoneal Mesothelial Cells；It is described
The step of (3) in colon cancer cell be colon cancer cell HCT116.
Preferably, the step (1) the specific steps are：
It is ready to be suspended in the fibroblast of culture solution, and is positioned in ice bath, type i collagen is added to 0.1mol/L
In NaOH, mixing immediately adds 10 × PBS or 10 × culture solution, mixing, and pH is 7 or so after mixing, and fibroblast is added
Suspension is added in culture vessel immediately after mixing.
In the external threedimensional model, fibroblastic number is 100~200 in every 100ul culture mediums.
Preferably, in the external threedimensional model, in fibroblast-type i collagen after the adherent stretching, extension of mesothelial cell
Complex surfaces form fused layer, and the compound body surface of fibroblast-type i collagen is completely covered in the fused layer
Face.
Preferably, the method carries out in gnotobasis.
Preferably, mesothelial cell's passage in fibroblast and the step (2) in the step (1) is not
It can be more than 10 generations.
Preferably, the mesothelial cell in fibroblast and the step (2) in the step (1) passes through shape
State, immunofluorescence technique identification.
Preferably, the colon cancer cell in the step (3) uses fluorescent marker.
Use the present invention for building the method that colon cancer cell peritonaeum shifts external threedimensional model, life can be simulated
Microenvironment is managed, the variation for more fully understanding adherency and invasive ability in colorectal cancer transfer process, it combines extracellular base
Matter, primary human world chrotoplast and primary people's peritoneal fibroblasts come study both matrix and extracellular matrix transfer process work
With, for study human world chrotoplast and human fibroblasts colorectal cancer cell Adhesion, Invasion this during effect,
And under co-culture system, whether cancer cell has normal cell the process of certain its canceration of promotion, will improve current in grinding
Study carefully the in-vitro cell culture model of colorectal cancer metastasis of cancer, and helps to enhance the totality to colorectal cancer early stage transfer process
Understand.
Description of the drawings
Fig. 1 is the primary human mesothelial cells cellular morphology that microscope white light shoots undertissue's separation and Extraction.
Fig. 2 is human fibroblasts' cellular morphology that microscope white light shoots undertissue's separation and Extraction.
Fig. 3 is that immunofluorescence technique identifies Peritoneal Mesothelial Cells：Keratin (Cytokeratin), vimentin
(Vimentin) antigen positive, prolyl hydroxylase (Prolyl-hydroxylase) are negative.
Fig. 4 is that immunofluorescence technique identifies peritoneal fibroblasts：Keratin (Cytokeratin), vimentin
(Vimentin), prolyl hydroxylase (Prolyl-hydroxylase) antigen positive.
Fig. 5 is the 3D model schematics established, and is mainly made of fibroblast, mesothelial cell and colon cancer cell,
Fibroblast is embedded in type i collagen, in the fused layer of top layer overlay mesothelial cell, and cancer cell is laid on most upper
Layer.
Fig. 6 is with cancer cell in HCT116 cell detection 2D and the 3D cultivating systems of fluorescent marker to the difference of drug susceptibility
It is different.
Fig. 7 is to carry out adhesive capacity measurement respectively in 2D and 3D culture models with the HCT116 cells of fluorescent marker, is led to
Cross the quantity that fluorescence microscope measures adherent cell.
Fig. 8 is to measure invasive ability respectively in 2D and 3D culture models with the HCT116 cells of fluorescent marker to measure, and is led to
Cross the quantity that fluorescence microscope measures intrusion cell.
Specific implementation mode
In order to more clearly describe the technology contents of the present invention, carried out with reference to specific embodiment further
Description.
The present invention includes step for building the method that colon cancer cell peritonaeum shifts external threedimensional model：
(1), fibroblast suspension is mixed with type i collagen, is added in culture vessel immediately after mixing；
(2), culture vessel is placed at room temperature 20 minutes, is subsequently placed in cell incubator after 2~4 hours and coagulates
Gu continuing to spread mesothelial cell in plate 18~38 hours；
(3) colon cancer cell is spread in plate, the structure of the outer 3D models of perfect aspect.
Wherein, the fibroblast in the step (1) is primary peritoneal fibroblasts；In the step (2)
Mesothelial cell be primary Peritoneal Mesothelial Cells.
The present invention provides a kind of Peritoneal Mesothelial Cells original cuiture, specifically includes：
1) the extraction separation of Peritoneal Mesothelial Cells
The relatively small number of peritoneal tissues of clip vascular fatty are placed in sterile training by the peritoneal tissues that sterile abdominal operation is extractd
It supports in ware, after being rinsed 3 times with sterile PBS, is cut into 3cm*3cm sizes with scissors, the peritoneal tissues of clip are placed in containing 500U/ml
Penicillin and 500ug/ml streptomysins PBS phosphate buffers in impregnate 20min, then with PBS rinse 5min, will rinse
Tissue afterwards is laid in sterile petri dish, then is rinsed repeatedly to no oil droplet and floated with PBS, while wiping out blood vessel and fat,
Clean tissue is cut into 1cm*1cm sizes, fritter is put into 50ml culture bottles, and 0.25% trypsase-EDTA cells are added
Digestive juice 20ml is incubated, and is positioned over 37 DEG C, 5%CO2The digested 25min of incubator, per 5min, oscillation is primary, and last 5min is kept
It is static.100 mesh stainless steels of digestive juice are sieved through filter in culture dish after digestion, addition equivalent contains 20% calf serum
RPMI1640 culture mediums terminate digestion.It is sub-packed in 10ml centrifuge tubes with pipettor, 1000r/min centrifuges 10min, abandons
Clearly, appropriate complete culture solution is added, cell precipitation is resuspended, be sub-packed in 50ml Tissue Culture Flasks, be positioned over 37 DEG C, volume fraction
For 5% CO2Culture in incubator.
2) Peritoneal Mesothelial Cells secondary culture
Primitive cell culture for 24 hours after, all outmoded culture solutions are replaced in bottle with isometric fresh complete culture solution, hereafter
Change liquid 1 time within every 3 days.About culture (4~7) d cell growths fusion, can pass on for the first time.When cell growth is fused into single layer, PBS
Rinsing 2 times, every bottle is added complete digestive juice 0.5ml, at 37 DEG C, 5%CO2, the digested 5min of incubator.Microscopic observation cell disappears
Change situation.After cell detachment, 5ml complete culture solutions are added and terminate digestion.Cell suspension is sub-packed in centrifuge tube, 1000r/ml
10min is centrifuged, supernatant is abandoned, appropriate complete culture solution is added, cell precipitation is resuspended, pass on respectively in 50ml culture bottles.It is added suitable
Measure complete culture solution, the stationary culture in incubator, until the completely adherent stretching, extension of cell.Mesothelial cell uses earlier generations (1~10
Generation) to reduce the change dedifferented with original table.
3) morphology, immunofluorescence technique is utilized to identify Peritoneal Mesothelial Cells
As shown in Figure 1, in cobblestone-appearance of paving the way under the Peritoneal Mesothelial Cells light microscopic being separately cultured；As shown in figure 3, immune group
Change identification and shows keratin (Cytokeratin), vimentin (Vimentin) antigen positive, prolyl hydroxylase (Prolyl-
Hydroxylase) feminine gender determines.It confirms that the cell of culture is PMC really, and excludes fibroblast, leucocyte, intravascular
Chrotoplast.
The present invention provides a kind of peritoneal fibroblasts original cuiture, specifically includes：
1) the extraction separation of peritoneal fibroblasts
Hyaluronidase and type III Collagenase of the peritoneal tissues containing 100U, total volume are the PBS of 100ml 37
DEG C digestion 6 hours.Tissue is abandoned, suspension containing cell is transferred in 15ml centrifuge tubes, and 1500rpm/min centrifuges 5min,
Precipitation is washed twice with RPMI1640 (containing 20% FBS and 100U/ml penicillin and 100ug/ml streptomysins) culture solution.At fiber
The purifying of cell is determined by the prolyl hydroxylase immunohistochemical staining positive.Fibroblast uses earlier generations (1~10 generation)
To reduce the change dedifferented with original phenotype.
2) peritoneal fibroblasts secondary culture
Primitive cell culture for 24 hours after, all outmoded culture solutions are replaced in bottle with isometric fresh complete culture solution, hereafter
Liquid is changed per 3d 1 time.About culture (4~7) d cell growths fusion, can pass on for the first time.When cell growth is fused into single layer, PBS drifts
Wash 2 times, every bottle is added complete digestive juice 0.5ml, at 37 DEG C, 5%CO2, the digested 2~5min of incubator.Microscopic observation cell
Digest situation.After cell detachment, 5ml complete culture solutions are added and terminate digestion.Cell suspension is sub-packed in centrifuge tube,
1000rpm/min centrifuges 10min, abandons supernatant, appropriate complete culture solution is added, cell precipitation is resuspended, pass on cultivated in 50ml respectively
In bottle.Appropriate complete culture solution is added, the stationary culture in incubator, until the completely adherent stretching, extension of cell.Before mesothelial cell uses
Several generations (1-10 generations) is to reduce the change dedifferented with original table.
3) morphology, immunofluorescence technique is used to identify peritoneal fibroblasts
As shown in Fig. 2, being in spindle shape under the peritoneal fibroblasts light microscopic being separately cultured；As shown in figure 4, immunohistochemistry is reflected
Surely keratin (Cytokeratin), vimentin (Vimentin) antigen positive, prolyl hydroxylase (Prolyl- are shown
Hydroxylase) positive determines.Confirm that the cell of culture is fibroblast really.
The present invention provides a kind of method of immunofluorescence technique identification of cell antigen presentation, specifically includes：
1) cell climbing sheet is fixed in culture plate, by mesothelial cell or fibroblast with RPMI1640 (contain 20%FBS,
100U/ml penicillin and 100ug/ml streptomysins) culture solution carries out bed board, after cell is adherent, embathe 3 times with PBS, every time
3min；
2) slide is embathed 3 times with 4% 4 DEG C of paraformaldehyde fixed creep plate 20min, PBS, each 3min；
3) 0.3%Triton X-100 (PBS preparations) the penetrating 20min of room temperature；
4) PBS embathes slide 3 times, and each 3min, blotting paper blots PBS, and 5%BSA (PBS preparations) is added dropwise on slide,
Room temperature closes 30min；
5) blotting paper sops up confining liquid, and the primary antibody (1 of sufficient amount diluted is added dropwise in every slide:50, PBS prepare, and contain
1%BSA and 0.1%Triton X-100) and it is put into wet box, 4 DEG C of overnight incubations；
6) add fluorescence secondary antibody：PBS embathes creep plate 3 times, each 3min, blotting paper blot be added dropwise after surplus liquid on creep plate it is dilute
The fluorescence secondary antibody released is incubated at least 1 hour for 20-37 DEG C in wet box, and PBS embathes slice 3 times, each 3min；Pay attention to.
7) core is redyed：DAPI (1 μ g/ml, PBS dilution) is added dropwise and is protected from light incubation 5min, dye core is carried out to sample, PBS is washed away
Extra DAPI is rinsed 3 times, each 3min；
8) image is acquired under the microscope in fluorescence microscopy.
The present invention provides a kind of embodiment of the outer three-dimensional metastasis model of construct, specifically includes：
Preparation containing fibroblastic three-dimensional collagen (to prepare 1 milliliter, for 1mg/ml three-dimensional glue)：It is ready to suspend
It in the fibroblast of culture solution, and is positioned in ice bath, 200ul rat tail collagen protein I types (5mg/ml) is added to
In 12ul0.1mol/L NaOH (if 12ul 0.1mol/L NaOH are added in collagen solution in turn, it can be due to NaOH not
Can rapid mixing and generate local collagen condensation), mixing, adds 10 × PBS of 23ul or 10 × culture solution immediately, mixing
(pH is 7 or so after mixing, if phenol red without adding in PBS or culture solution, needs to be tested with pH test paper when first used)；
The fibroblast suspension of 760ul is added, is added in culture vessel immediately after mixing.By culture vessel in room temperature
Lower placement solidifies after 20 minutes, 2~4 hours, continues to spread mesothelial cell in plate.Mesothelial cell is in 1ml culture medium middle berths
Enter in plate after 18~38 hours, HCT116-GFP cells is spread to complete the structure that 3D cultivates jellium model in plate, Fig. 5 is shown
3D model schematics provided by the invention.
Embodiment 1.MTT experiments
MIT experiment for detection fluorescent marker HCT116 cells in 3D cultivating systems relative to the drug in 2D cultivating systems
Sensibility.
In 96 well culture plates：(RPMI1640 contains 200 fibroblast mixing rat tail collagen protein I types in the medium
The streptomysin of 20%FBS, 100U/ml penicillin and 100ug/ml).After gelling is solid, 10000 primary mesothelial cells are added to training
In the system of supporting, 37 DEG C of incubations converge layer (18~38 hours) until mesothelial cell forms one.HCT116-GFP cell suspensions connect
Kind is in every hole.After cell is completely adherent, anticarcinogen 5-FU is administered, and after being administered 24 hours, passes through fluorescence microscope 100
Times mirror obtains, and quantitative cell survival number calculates cell survival rate, three repeated experiments.
As shown in fig. 6, compared to 2D cultivating systems, the colon cancer cell under 3D model systems in the present invention is in embodiment
In 1, shows stablizing for the stronger certain drug resistance for meeting internal colon cancer development trend and keep.
2. adhesion experiment of embodiment
Adhesion experiment be detect fluorescent marker HCT116 cell adherences to mesothelial cell and/or wrap up fibroblastic I
The ability of Collagen Type VI fused layer.
In 96 well culture plates：(RPMI1640 contains 200 fibroblast mixing rat tail collagen protein I types in the medium
The streptomysin of 20%FBS, 100U/ml penicillin and 100ug/ml).After gelling is solid, 10000 primary mesothelial cells are added to training
In the system of supporting, 37 DEG C of incubations converge layer (18~38 hours) until mesothelial cell forms one.HCT116-GFP cell suspensions connect
Kind is in every hole.Adherent cell is obtained by 100 times of mirrors of fluorescence microscope, the cell number quantitatively adhered to.Three repeated experiments.
As shown in fig. 7, compared to 2D cultivating systems, the colon cancer cell under 3D model systems in the present invention is in embodiment
In 2, the cell adherence ability of the stronger enhancing for meeting internal colon cancer development process is shown.
3. Matrigel of embodiment
Matrigel is the HCT116 cellular infiltrations of detection fluorescent marker to mesothelial cell and wraps up fibroblastic I type
The locomitivity of collagen fusion layer.
One layer is first covered in Transwell (model in the apertures 8um) cell of 24 well culture plates contains 200 fibroblasts
With the mixture (50ul culture mediums) of I type of rat tail collagen protein, it is incubated 2-4 hours at 37 DEG C.Containing 10000 mesothelial cells
100ul culture solutions be added to I type of rat tail collagen protein and fibroblast mixed gel surface, at 37 DEG C cultivate 18-38 it is small
When.HCT116-GFP cell suspension inoculations are in every hole.Cell is obtained by 100 times of mirrors of fluorescence microscope, quantitatively penetrates cell
Reach the cell number of cell lower part.Three repeated experiments.
As shown in figure 8, compared to 2D cultivating systems, the colon cancer cell under 3D model systems in the present invention is in embodiment
In 3, the locomitivity of similar colon cancer cell is shown.
Peritoneal Mesothelial Cells are to constitute the main cell group of peritonaeum, and can secrete cytokine profiles, are that peritonaeum is primary
The parenchyma of tumour, also being played in peritoneal fibrosiss and abdominal cavity are adhered makes and key effect.Fibroblast includes cancer
Matrix major cellular component.Some researches show that cancer associated fibroblast cells, and the non-tumorigenic of starting can be stimulated normal
The tumour progression of epithelial cell and the growth for promoting colon cancer in animal model.Meanwhile other scientific research personnel have found that colon cancer is thin
The interaction of born of the same parents and stroma cell can activate the β-catenin signal paths in cancer cell and lead to colon cancer stem cell
Increase.Therefore, understand the molecular mechanism that paracrine interacts between tumour and surrounding substrate to will be helpful to have found that it is likely that as latent
In the recruit of drug target.
When monolayer cell culture (2D) scale-model investigation tumours of chemotherapeutic sensibility, obtained result often has with experiment in vivo
Great gap.It is cultivated compared to 2D, 3D cultivates the growing state that can simulate cancerous tissue well in vitro, including invasion
Property, drug resistance, changes in gene expression and protein secretion situation.The present invention is directed to build tumor formation situation and cancer cell in analogue body to see
Interaction, especially the dimensional culture model of intercellular adhesion.From the tumour cell with drug resistance isolated in vivo
When carrying out cultured in monolayer in vitro, drug resistance mostly occurs reduction or disappearance；And in Three-dimensional cell culture model, cell gradually has
Standby drug resistance.Peritonaeum transfer threedimensional model (mesothelial cell, fibroblast, colon cancer cancer cell) come study colon cancer adherency and
Invasion.3D models include the primary fibroblast obtained from peritonaeum, are mixed with type i collagen, then cover one layer and obtained from peritonaeum
The primary mesothelial cell taken.After adding colon cancer cell, the microcosmic metastasis model of peritonaeum of dimensional culture is built.3D culture or
The cell grown in three-dimensional aggregates (multicellular spheriods) has preferably reappeared the internal structure of tissue and tumour.3D culture systems
It can solve the human tumour in various cancers model and the functional interaction between stroma cell.
Use the present invention for building the method that colon cancer cell peritonaeum shifts external threedimensional model, life can be simulated
Microenvironment is managed, the variation for more fully understanding adherency and invasive ability in colorectal cancer transfer process, it combines extracellular base
Matter, primary human world chrotoplast and primary people's peritoneal fibroblasts come study both matrix and extracellular matrix transfer process work
With, for study human world chrotoplast and human fibroblasts colorectal cancer cell Adhesion, Invasion this during effect,
And under co-culture system, whether cancer cell has normal cell the process of certain its canceration of promotion, will improve current in grinding
Study carefully the in-vitro cell culture model of colorectal cancer metastasis of cancer, and helps to enhance the totality to colorectal cancer early stage transfer process
Understand.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that can still make
Various modifications and alterations are without departing from the spirit and scope of the invention.Therefore, the description and the appended drawings should be considered as illustrative
And not restrictive.
Claims (9)
1. a kind of for building the method that colon cancer cell peritonaeum shifts external threedimensional model, which is characterized in that the method
Including step：
(1), fibroblast suspension is mixed with type i collagen, is added in culture vessel immediately after mixing；
(2), culture vessel is placed at room temperature 20 minutes, is subsequently placed in cell incubator and is solidified after 2~4 hours, after
It is continuous to spread mesothelial cell in plate 18~38 hours；
(3) colon cancer cell is spread in plate, the structure of the outer 3D models of perfect aspect.
2. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
It is, the fibroblast in the step (1) is primary peritoneal fibroblasts；Type i collagen in the step (1)
For I type rat tail collagen proteins；Mesothelial cell in the step (2) is primary Peritoneal Mesothelial Cells；In the step (3)
Colon cancer cell be colon cancer cell HCT116.
3. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
Be, the step (1) the specific steps are：
It is ready to be suspended in the fibroblast of culture solution, and is positioned in ice bath, type i collagen is added to 0.1mol/L NaOH
In, mixing immediately adds 10 × PBS or 10 × culture solution, mixing, and pH is 7 after mixing, and fibroblast suspension is added,
It is added in culture vessel immediately after mixing.
4. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
It is, in the external threedimensional model, fibroblastic number is 100~200 in every 100ul culture mediums.
5. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
It is, in the external threedimensional model, in fibroblast-type i collagen complex surfaces after the adherent stretching, extension of mesothelial cell
Fused layer is formed, and the fibroblast-type i collagen complex surfaces are completely covered in the fused layer.
6. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
It is, the method carries out in gnotobasis.
7. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
It is, mesothelial cell's passage in the fibroblast and the step (2) in the step (1) may not exceed 10
Generation.
8. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
It is, the mesothelial cell in fibroblast and the step (2) in the step (1) is by morphology, immune
Fluorescence method is identified.
9. according to claim 1 for building the method that colon cancer cell peritonaeum shifts external threedimensional model, feature
It is, the colon cancer cell in the step (3) uses fluorescent marker.
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