CN110129262A - A kind of tumour cell co-cultures threedimensional model and its construction method and application - Google Patents
A kind of tumour cell co-cultures threedimensional model and its construction method and application Download PDFInfo
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- 235000010443 alginic acid Nutrition 0.000 claims abstract description 19
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- 238000012216 screening Methods 0.000 claims abstract description 7
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- 229940005550 sodium alginate Drugs 0.000 claims description 21
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical group CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 14
- 235000010413 sodium alginate Nutrition 0.000 claims description 14
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical group [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 238000011156 evaluation Methods 0.000 claims description 9
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- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to tumour cell dimensional culture technical fields more particularly to a kind of tumour cell to co-culture threedimensional model and its construction method and application.The present invention carries out crosslinking using alginate and calcium salt and prepares microballoon as cytoskeleton, the anchor point of cell attachment is provided, good three dimensional growth environment can be provided for cell, and, the present invention co-cultures tumour cell and fibroblast three-dimensional, endothelial cell and cell mixing microballoon are carried out by cell co-cultivation by Transwell device again, building is three-dimensional to co-culture tumor-vascular external model, tumour cell strengthens cell-ECM under the conditions of three-dimensional co-cultures, interaction between cell-matrix, make tumour cell that there is broader growing space, the living environment of tumour cell in vivo can more accurately be simulated, so that the tumour cell threedimensional model of building is applied to the screening of anti-tumor drug and/or evaluates more accurate, reliably, it can reduce the cost of anti-tumor drug research and development.
Description
Technical field
The invention belongs to tumour cell dimensional culture technical fields more particularly to a kind of tumour cell to co-culture threedimensional model
And its construction method and application.
Background technique
Tumor model is most important for cancer drug development, and conventional two-dimensional cell model structure is single, cell growth
Condition is limited, and cell-cell interaction is faint, and tumour agglomerating can not be grown, and is not easy to shift or invade, it is difficult in accurate simulation body
The complex environment of tumour, the drug evaluation and/or screening carried out using two-dimentional cell model is inaccurate.
Therefore, building one more acurrate can simulate human tumor growing environment and the model substitution two dimension of economical and efficient is thin
Born of the same parents' model for drug development and evaluates and screens most important.
Summary of the invention
In view of this, the present invention provides a kind of tumour cells to co-culture threedimensional model and its construction method and application, use
It being capable of the more acurrate model for simulating human tumor growing environment and economical and efficient in building one.
The specific technical solution of the present invention is as follows:
A kind of tumour cell co-cultures the construction method of threedimensional model, comprising the following steps:
A) tumour cell and fibroblast are configured to cell mixing suspension, added in the cell mixing suspension
Enter alginate solution, obtains cell mixing-alginate solution；
B) cell mixing-alginate solution drop is obtained into cell mixing microballoon in calcium salt soln；
C) after endothelial cell being seeded to Transwell device upper layer cell, the cell mixing microballoon is seeded to
Transwell device bottom chamber carries out cell culture, obtains tumour cell and co-cultures threedimensional model.
Preferably, tumour cell described in the cell mixing suspension and fibroblastic quantitative proportion are (2
~3): (1~2).
Preferably, concentration of the tumour cell in the cell mixing suspension is 1*105A/ml~5*106A/
ml；
Concentration of the fibroblast in the cell mixing suspension is 1*105A/ml~5*106A/ml.
Preferably, it is thin to be selected from prostate gland cancer cell, breast cancer cell, Leiomyoma Cell or liver cancer for the tumour cell
Born of the same parents；
The endothelial cell is selected from Human umbilical vein endothelial cells, vein endothelial cell or arterial endothelium cells.
Preferably, the alginate is sodium alginate；
The calcium salt is selected from calcium chloride or calcium sulfate.
Preferably, the mass volume ratio concentration of the alginate solution is 2%~4%；
The mass ratio of the cell mixing suspension and the alginate solution is (1~3): (1~3).
Preferably, the concentration of the calcium salt soln is 0.1~0.2mol/L.
Preferably, the tumour cell of the cell mixing microballoon and fibroblast are dense in the inoculation of Transwell device
Degree is respectively 1*105A/ml~5*106A/ml and 1*105A/ml~5*106A/ml.
The tumour cell obtained the present invention also provides construction method described in above-mentioned technical proposal co-cultures threedimensional model.
The present invention also provides tumour cells described in above-mentioned technical proposal to co-culture threedimensional model in screening anti-tumor medicine
And/or the application in evaluation.
In conclusion the present invention provides the construction method that a kind of tumour cell co-cultures threedimensional model, including following step
It is rapid: tumour cell and fibroblast a) being configured to cell mixing suspension, sea is added in the cell mixing suspension
Alginate soln obtains cell mixing-alginate solution；B) cell mixing-alginate solution drop is molten in calcium salt
In liquid, cell mixing microballoon is obtained；C) the cell mixing microballoon is seeded to Transwell device bottom chamber, it is described interior
Chrotoplast is seeded to Transwell device upper layer cell, carries out cell culture, obtains tumour cell and co-cultures threedimensional model.This
In invention, crosslinking is carried out using alginate and calcium salt and prepares microballoon as cytoskeleton, the anchor point of cell attachment is provided, is reduced
Tumour cell carries out the cost of dimensional culture, and alginate has good biocompatibility and non-immunogenic, a Neng Gouwei
Cell provides good three dimensional growth environment, also, the present invention co-cultures tumour cell and fibroblast three-dimensional, then passes through
Endothelial cell and cell mixing microballoon are carried out cell co-cultivation by Transwell device, construct three-dimensional co-cultivation tumor-vascular body
External model, tumour cell strengthen the interaction between cell-ECM, cell-matrix under the conditions of three-dimensional co-cultures, make to swell
Oncocyte has broader growing space, can more accurately simulate the living environment of tumour cell in vivo, so that building
Tumour cell threedimensional model be applied to anti-tumor drug screening and/or evaluation it is more accurate, reliable, can reduce antitumor
The cost of medicament research and development.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is that a kind of tumour cell provided in the embodiment of the present invention co-cultures the schematic diagram of threedimensional model；
Fig. 2 be in the embodiment of the present invention 1 cell mixing microballoon on day 4, the 8th day, the survival rate of the 10th day and the 12nd day.
Specific embodiment
The present invention provides a kind of tumour cells to co-culture threedimensional model and its construction method and application, for constructing one
It being capable of the more acurrate model for simulating human tumor growing environment and economical and efficient.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Referring to Fig. 1, a kind of tumour cell to provide in the embodiment of the present invention co-cultures the schematic diagram of threedimensional model.
A kind of tumour cell co-cultures the construction method of threedimensional model, comprising the following steps:
A) tumour cell and fibroblast are configured to cell mixing suspension, sea is added in cell mixing suspension
Alginate soln obtains cell mixing-alginate solution；
B) cell mixing-alginate solution drop is obtained into cell mixing microballoon in calcium salt soln；
C) after endothelial cell being seeded to Transwell device upper layer cell, the cell mixing microballoon is seeded to
Transwell device bottom chamber carries out cell culture, obtains tumour cell and co-cultures threedimensional model.
In the present invention, crosslinking is carried out using alginate and calcium salt and prepares microballoon as cytoskeleton, cell attachment is provided
Anchor point, reduce the cost that tumour cell carries out dimensional culture, alginate has good biocompatibility and nonimmune
Originality can provide good three dimensional growth environment for cell, also, tumour cell and fibroblast three-dimensional are total to by the present invention
Culture, then endothelial cell and cell mixing microballoon are carried out by cell co-cultivation by Transwell device, construct three-dimensional co-culture
Tumor-vascular external model, tumour cell strengthen the phase between cell-ECM, cell-matrix under the conditions of three-dimensional co-cultures
Interaction makes tumour cell have broader growing space, can more accurately simulate the existence ring of tumour cell in vivo
Border, so that the tumour cell threedimensional model of building is applied to the screening of anti-tumor drug and/or evaluates more accurate, reliable, energy
Enough reduce the cost of anti-tumor drug research and development.
The embodiment of the present invention co-cultures tumour cell and fibroblast in calcium alginate microsphere, then passes through
Cell mixing microballoon and endothelial cell are carried out cell co-cultivation by Transwell device, and building one being capable of more acurrate analogue body
The tumour cell threedimensional model of interior tumour growth microenvironment.The embodiment of the present invention is by drug evaluation to the tumour cell three-dimensional mould
Type is verified, and is compared and analyzed with conventional two-dimensional cell model, the results showed that tumour cell of the embodiment of the present invention is three-dimensional
Being successfully established for model is expected to evaluate for anticancer drug and new drug output is promoted to provide reliable technical guarantee.
In the embodiment of the present invention, in cell mixing suspension tumour cell and fibroblastic quantitative proportion be (2~
3): (1~2), preferably 2:1.
In the embodiment of the present invention, concentration of the tumour cell in cell mixing suspension is 1*105A/ml~5*106A/
Ml, preferably 1*106A/ml；
Concentration of the fibroblast in cell mixing suspension is 1*105A/ml~5*106A/ml, preferably 5*105
A/ml.
In the embodiment of the present invention, tumour cell is selected from prostate gland cancer cell, breast cancer cell, Leiomyoma Cell or liver cancer
Cell.
In the embodiment of the present invention, need to choose and swell according to the composed structure of tumour growing environment in vivo, tumour
The cell that oncocyte co-cultures, needs growth, division cycle and the cancer cell of the cell close, and condition of culture is similar (available same
Kind culture medium culture).Tumour cell is preferably prostate gland cancer cell, the growth of prostate gland cancer cell, division cycle at fiber
The difference very little of cell, the condition of cell culture is close, can use culture medium culture of the same race.Also, the growth of prostate gland cancer cell
Speed is fast, to the of less demanding of growing environment, is easy to cultivate, is suitble to the short time to cultivate a large amount of cell and is tested.
Endothelial cell is selected from Human umbilical vein endothelial cells, vein endothelial cell or arterial endothelium cells.In people
In in-vivo tumour environment, tumour have the blood vessel of very abundant it is interspersed, around wherein, and endothelial cell be to be formed it is tumor vascular
A kind of important cells have important role to tumour growth metabolism, in order to preferably simulate the growing environment of tumour, this
Application co-cultures endothelial cell and tumour cell.
Prostate gland cancer cell is selected from PC3, LNCaP, DU145, VCaP or VLcop；Fibroblast be selected from 3T3, BMF,
HPF, HFF or BJ.
In the embodiment of the present invention, alginate is sodium alginate, and sodium alginate is a kind of to be extracted from the natural of algae
Polysaccharide, it is at low cost, be easy to make；
Calcium salt is selected from calcium chloride or calcium sulfate.
In the embodiment of the present invention, the mass volume ratio concentration of alginate solution is 2%~4%, preferably 3.5%, real
It tests the result shows that cell is in the bead that sodium alginate mass volume ratio concentration is 3.5%, survival rate is higher, more stable.
The mass ratio of cell mixing suspension and alginate solution is (1~3): (1~3), preferably 1:1.
The molecular weight of alginate is preferably 216.12303.
In the embodiment of the present invention, the concentration of calcium salt soln is 0.1~0.2mol/L, preferably 0.1mol/L.
In the embodiment of the present invention, before step a), further includes: tumour cell and fibroblast are carried out plane training respectively
It supports.
Step a) tumour cell is the tumour cell in the 3rd generation to the 6th generation, and fibroblast is the 3rd generation to the 6th generation into fibre
Tie up cell.
In the embodiment of the present invention, the tumour cell of the cell mixing microballoon and fibroblast are in Transwell device
Inoculum density be respectively 1*105A/ml~5*106A/ml and 1*105A/ml~5*106A/ml.
In the embodiment of the present invention, endothelial cell is seeded to behind the cell of Transwell device upper layer for 24 hours by step c), will be mixed
It closes cell microsphere and is seeded to Transwell device bottom chamber.It is small that Transwell device lower layer is seeded in cell mixing microballoon
Before room, endothelial cell is in Transwell device upper layer cell adherent growth and reaches stable state.
The tumour cell obtained the embodiment of the invention also provides above-mentioned technical proposal construction method co-cultures threedimensional model.
Threedimensional model is co-cultured the embodiment of the invention also provides above-mentioned technical proposal tumour cell to sieve in anti-tumor drug
Application in choosing and/or evaluation.
Tumour cell threedimensional model of the embodiment of the present invention is set up based on growth of cancer cells environment in human body, with
The cancer cell cultivated under two-dimensional condition shows larger difference in all fields, can be to internal in tumour cell threedimensional model
Efficacy of drugs has better predictability.Tumour cell of embodiment of the present invention threedimensional model can also carry out drug toxicity test, lead to
Different evaluation means are crossed to carry out multi-angle evaluation to drug antitumaous effect.
Tumour cell threedimensional model of the embodiment of the present invention reduces experiment using easy to operate, experimental situation requirement is lower
A possibility that fault, and be applied in the screening and/or evaluation of anti-tumor drug accurately, reliably, can reduce antineoplastic
The cost of object research and development.
Tumour cell threedimensional model of the embodiment of the present invention can be applied to the screening and detection of new anticancer drug, due to cell
Culture is convenient to take out detection at any time in the microballoon that calcium alginate is formed, to study different role time drug to tumour cell
Function and effect.Tumour cell of embodiment of the present invention threedimensional model can also be createed by the diaphragm in replacement Transwell
New model, or micro fluidic device is combined, model is innovated to construct the tumour cell of new more bionical effect
Model.
For a further understanding of the present invention, the present invention will be described in detail combined with specific embodiments below.
Embodiment 1
The present embodiment carries out the preparation that tumour cell co-cultures cell mixing microballoon in threedimensional model, comprising the following steps:
1) preparation of sodium alginate soln and calcium chloride solution
It weighs sodium alginate powder and is added in reagent bottle and is added deionized water, be then placed in magnetic stir bar, be placed on
12h~stir for 24 hours is carried out on magnetic stirring apparatus, prepare obtain mass concentration be respectively 3%, 3.5% and 4% sodium alginate it is molten
Liquid.
It weighs calcium chloride powder and is added in reagent bottle and is added deionized water, the calcium chloride that preparation obtains 0.1mol/L is molten
Liquid.
Sodium alginate soln and calcium chloride solution are put into togerther in autoclave sterilization machine and carry out 125 DEG C of 15 points of sterilizings
Clock, sodium alginate soln and calcium chloride solution, which are prepared, to be completed.
2) the plane culture of prostate gland cancer cell (PC3) and fibroblast (3T3)
The cryovial that PC3 cell is housed is taken out, water-bath is melted, centrifugation, with containing 1% dual anti-, 10% fetal calf serum low sugar
DMEM culture solution culture PC3 cell, changes a not good liquor in every 2 days.When cell confluent cultures bottom of bottle 75%-80%, trypsase is used
It is digested, it is in ellipticity that PC3 cell to PC3 cell appearance is observed in microscope, i.e., when separating de- wall, terminates digestion.
It takes PC3 cell suspension to be centrifuged, abandons supernatant, the culture solution that proper amount of fresh is added is resuspended, and being made into cell concentration is (1*106A/
Ml PC3 cell suspending liquid).
The cryovial that 3T3 cell is housed is taken out, water-bath is melted, centrifugation, with containing 1% dual anti-, 10% fetal calf serum low sugar
DMEM culture solution culture 3T3 cell, changes a not good liquor in every 2 days.When cell is paved with bottom of bottle 75%-80%, carried out with trypsase
Digestion, it is in ellipticity that 3T3 cell to 3T3 cell appearance is observed in microscope, i.e., when separating de- wall, terminates digestion.Take 3T3
Cell suspension centrifugation, abandons supernatant, and the culture solution that proper amount of fresh is added is resuspended, and obtains 3T3 cell suspending liquid, and 3T3 cell suspends
The cell concentration of liquid is consistent with the cell concentration of PC3 cell suspending liquid.
3) cell mixing microballoon is prepared
PC3 cell suspending liquid and 3T3 cell suspending liquid are added with 1:1 ratio into centrifuge tube, it is dense to be configured to total cell
Degree is 1 × 106Then the cell mixing suspension of/mL takes three centrifuge tubes, be separately added into mass concentration thereto and be respectively
3%, 3.5% and 4% sodium alginate soln, then with sodium alginate soln: it is thin that mixing is added in cell mixing suspension liquid proportional 1:1
Then solution is blown and beaten uniformly with liquid-transfering gun, obtains cell mixing-sodium alginate soln 1, cell mixing-seaweed by born of the same parents' suspension
Acid sodium solution 2 and cell mixing-sodium alginate soln 3.
Culture dish is taken, the equivalent calcium chloride solution of 0.1mol/L is added, draws cell mixing-seaweed respectively using syringe
Syringe needle is suspended on training by acid sodium solution 1, cell mixing-sodium alginate soln 2 and cell mixing-sodium alginate soln 3
In feeding ware more than calcium chloride liquid level, solution is slowly oozed with 1 drop/sec of speed, makes cell mixing-sodium alginate soln 1, mix
It closes cell-sodium alginate soln 2 and cell mixing-sodium alginate soln 3 and calcium chloride solution is cross-linked to form cell mixing respectively
Microballoon 1, cell mixing microballoon 2 and cell mixing microballoon 3, then by cell mixing microballoon 1, cell mixing microballoon 2 and cell mixing
Microballoon 3 is incubated at fresh contain in 1% dual anti-, 10% fetal calf serum low sugar DMEM culture solution.Wherein, cell mixing microballoon 1,
Cell mixing microballoon 2 and cell mixing microballoon 3 are all provided with 5 groups of parallel tests.
By cell mixing microballoon 1, cell mixing microballoon 2 and cell mixing microballoon 3 respectively the 0th day, the 4th day, the 8th day,
It carries out cell collection within 10th day and the 12nd day and measures the survival rate of cell, be in terms of 100% by the 0th day cell survival rate.It please join
Read Fig. 1, be in the embodiment of the present invention 1 cell mixing microballoon on day 4, the 8th day, the survival rate of the 10th day and the 12nd day, as a result
Show the cell survival rate highest for the cell mixing microballoon for using mass concentration to obtain for 3.5% sodium alginate.
Embodiment 2
The present embodiment carries out the building that tumour cell co-cultures threedimensional model with 1 method of embodiment.
Cell mixing microballoon is obtained with 1 method of embodiment, wherein the mass concentration of sodium alginate is 3.5%.
With 7.2 × 104The cell density of a/ml is in Transwell device upper layer cell culture Human umbilical vein endothelial cells
(HUVECs), specifically: HUVECs is cultivated in two-dimensional environment to the third generation, HUVECs is collected by centrifugation and is resuspended in culture medium
In, it is inoculated into the small indoor culture in Transwell device upper layer, culture solution is containing 1% dual anti-, 10% fetal calf serum low sugar
DMEM.After one day, it will co-culture, mix in orifice plate that cell mixing microballoon is inoculated in Transwell device bottom chamber
Fibroblastic inoculum density of the tumour cell and cell mixing microballoon that close cell microsphere is respectively 1*106A/ml and 5*
105A/ml, changes liquid every other day, obtains tumour cell and co-cultures threedimensional model.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. the construction method that a kind of tumour cell co-cultures threedimensional model, which comprises the following steps:
A) tumour cell and fibroblast are configured to cell mixing suspension, sea is added in the cell mixing suspension
Alginate soln obtains cell mixing-alginate solution；
B) cell mixing-alginate solution drop is obtained into cell mixing microballoon in calcium salt soln；
C) after endothelial cell being seeded to Transwell device upper layer cell, the cell mixing microballoon is seeded to
Transwell device bottom chamber carries out cell culture, obtains tumour cell and co-cultures threedimensional model.
2. construction method according to claim 1, which is characterized in that tumour cell described in the cell mixing suspension
It is (2~3): (1~2) with fibroblastic quantitative proportion.
3. construction method according to claim 2, which is characterized in that the tumour cell is in the cell mixing suspension
In concentration be 1*105A/ml~5*106A/ml；
Concentration of the fibroblast in the cell mixing suspension is 1*105A/ml~5*106A/ml.
4. construction method according to claim 1, which is characterized in that the tumour cell is selected from prostate gland cancer cell, cream
Adenocarcinoma cell, Leiomyoma Cell or liver cancer cells；
The endothelial cell is selected from Human umbilical vein endothelial cells, vein endothelial cell or arterial endothelium cells.
5. construction method according to claim 1, which is characterized in that the alginate is sodium alginate；
The calcium salt is selected from calcium chloride or calcium sulfate.
6. construction method according to claim 1, which is characterized in that the mass volume ratio concentration of the alginate solution
It is 2%~4%；
The mass ratio of the cell mixing suspension and the alginate solution is (1~3): (1~3).
7. construction method according to claim 1, which is characterized in that the concentration of the calcium salt soln is 0.1~0.2mol/
L。
8. construction method according to claim 1, which is characterized in that the tumour cell of the cell mixing microballoon and at fibre
It is respectively 1*10 that cell, which is tieed up, in the inoculum density of Transwell device5A/ml~5*106A/ml and 1*105A/ml~5*106
A/ml.
9. the tumour cell that construction method described in claim 1 to 8 any one obtains co-cultures threedimensional model.
10. tumour cell described in claim 9 co-cultures application of the threedimensional model in screening anti-tumor medicine and/or evaluation.
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