CN108998441A - A kind of three-dimensional nodule ball culture medium additive, culture medium and three-dimensional nodule ball cultural method - Google Patents
A kind of three-dimensional nodule ball culture medium additive, culture medium and three-dimensional nodule ball cultural method Download PDFInfo
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- CN108998441A CN108998441A CN201810868902.5A CN201810868902A CN108998441A CN 108998441 A CN108998441 A CN 108998441A CN 201810868902 A CN201810868902 A CN 201810868902A CN 108998441 A CN108998441 A CN 108998441A
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Abstract
The present invention provides a kind of three-dimensional nodule ball culture medium additive, culture medium and three-dimensional nodule ball cultural methods.The additive by thickener and glucosamine gel group at, thickener is selected from one of methylcellulose, ethyl cellulose, basement membrane matrix, collagen or a variety of combinations, and gelling agent, which is selected from, ties one of cold foot, alginic acid or Ago-Gel or a variety of combinations.The culture medium of the three-dimensional nodule ball, including tumor cell culture base and three-dimensional nodule ball culture medium additive above-mentioned.Culture medium of the present invention can be in High Density Cultivation as the result is shown for various kinds of cell system, hence it is evident that reduces the aggregation of tumour ball, fusion.
Description
Technical field
Invention is related to biological field, and in particular to a kind of culture medium for preparing tumour ball and its tumour ball are a large amount of three-dimensional (3D)
The method of preparation.
Background technique
Tumor stem cell (Cancer stem cells) theory thinks have a group to have stem cell properties in tumor tissues
Tumour cell, they are only the basic reason of tumorigenesis, Preventive.Tumour ball culture is presently the most common
The method of culture and enrichment tumor stem cell, identification of cell stemness ability.It is all in serum-free and (the ultralow adherency of non-adherent environment
Ware) under cultivated, through culture in one week or so is part of there is " stemness " characteristic cell to form tumour ball.Culture scheme has more
Kind, such as classical DMEM/F12+20ng/ml EGF+20ng/ml FGF+2%B27 scheme.Specifically added ingredient includes:
Epidermal growth factor (EGF), fibroblast growth factor (FGF), leukocyte inhibitory factor (LIF), B27, insulin, conversion
Growth factor-beta (TGF-β) etc..
Traditional tumour ball cultivation used by the prior art is easy to happen aggregation, adherency, agglomerating, loses tumour ball list
The feature of cell origin, and tumour ball culture amount is few, costly, operation difficulty is big.
Summary of the invention
It is an advantage of the invention to provide a kind of three-dimensional (3D) tumour ball culture medium additives.
Second object of the present invention is, provides a kind of three-dimensional nodule ball culture medium.
Third object of the present invention is, provides a kind of three-dimensional nodule ball cultural method.
According to an aspect of the present invention, a kind of three-dimensional nodule ball culture medium additive is provided, by thickener and gel
Agent composition；
Inventor thinks in conventional cell suspension medium, due under non-adherent environment, without attachment point in cell growth,
Will occur under extraneous micro force mobile and then lead to aggregation, adhere to, is agglomerating.Since tumour ball is easy aggregation, adherency
Therefore often by reducing cell inoculation quantity come the less above phenomenon in actual application；This results in the tumour ball amount of culture
It is fewer.And the present invention is found surprisingly that and can be achieved with high density, big by the way that two kinds of biomaterials of thickener and gelling agent are added
The purpose of amount culture tumour ball.
Thickener can increase culture medium viscosity to limit movement of the tumour ball in all directions, and gelling agent, which has, promotees gel
Effect makes culture medium generate certain degree of hardness and then controls the sinking speed of tumour ball, generates the effect of 3D culture.
Further, thickener is in methylcellulose (MC), ethyl cellulose, basement membrane matrix, collagen
One or more combinations；Gelling agent is selected from one of gellan gum, alginic acid or Ago-Gel or a variety of combinations；
Still further, the thickener and the mass ratio of gelling agent are 1-100:1, preferably 5-15:1, more preferably
For 10:1；
When using additive, the mass volume ratio of the thickener and culture medium is 0.1-0.5%, preferably 0.3%；It is solidifying
The mass volume ratio of jelly and culture medium be 0.01%-0.05%, preferably 0.03%.
According to a further aspect of the invention, a kind of culture medium of three-dimensional nodule ball is provided comprising tumour cell training
Base and three-dimensional nodule ball culture medium additive above-mentioned are supported, the tumor cell culture base is selected from arbitrary cell commonly used in the art
Culture medium, preferably serum-free tumour ball culture medium；
Further, the culture medium of three-dimensional nodule ball of the invention be in DMEM/F12 be added EGF, FGF, B27,
0.3% methylcellulose (MC) and the cold foot of 0.03% knot (GG) is made；
Still further, the culture medium of three-dimensional nodule ball of the invention be in DMEM/F12 be added 20ng/ml EG F,
The FGF of 20ng/ml, 2% B27 (adding commodity B27 solution according to volume 1:50), 0.3% methylcellulose (MC) and
0.03% gellan gum (GG) is made；
The preparation method of three-dimensional nodule ball culture medium of the present invention includes the following steps:
1) it prepares three-dimensional nodule ball culture medium additive: thickener and gelling agent being dissolved with water respectively, are configured to sterile
Aqueous thickener solution and gelling agent aqueous solution；
2) culture medium for the culture three-dimensional nodule ball that suspends is prepared: aqueous thickener solution, gelling agent aqueous solution and tumour is thin
The mixing of born of the same parents' culture medium obtains suspending and cultivates the culture medium of three-dimensional nodule ball.
Wherein, the thickener in step 1) is formulated as 0.1g/ml-0.5g/ml (preferably 0.3g/ml), and gelling agent is prepared
For 0.01g/ml-0.05g/ml (preferably 0.03g/ml).
According to a further aspect of the invention, the method for three-dimensional nodule ball culture is provided comprising following steps:
Tumour cell is digested and is counted by I, and the tumor cell inoculation digested is arrived aforementioned three-dimensional nodule ball to kind and is trained
It supports in base,
Centrifugal process replaces culture medium after II culture tumour cell turns yellow to culture medium, cultivates thereafter to the complete shape of tumour ball
At；
Wherein, the tumor cell density digested described in step I is 103-105Cells/ml is significantly higher than traditional culture
Base is inoculated with quantity (103cells/ml)；
Wherein, cell culture is in culture bottle or culture dish, condition of culture, and 37 DEG C, 5%CO2。
Wherein, culture medium process is replaced are as follows:
1) culture medium containing tumour ball is collected into centrifuge tube, and isometric DMEM solution is added and is diluted；
2) centrifuge is centrifuged；
3) old culture medium is removed, new culture medium is added.
Wherein, the diameter of the tumour ball of acquisition is more than 100 microns.
The utility model has the advantages that
Culture medium additive of the invention can change conventional medium property, and to the observation of tumour ball, counting, statistics, knot
Fruit shows that new technology can obviously reduce the aggregation of tumour ball, adherency, fusion, and high density, mass propgation are realized in culture bottle, real
Now to the enrichment culture of tumor stem cell, and the cell stemness of tumour ball is not reduced, obtain in various kinds of cell system good
Effect.
Detailed description of the invention
Influence of Fig. 1 MC to tumour ball aggregation fusion in culture medium
The influence that Fig. 2 GG settles tumour ball in culture medium
5-8F tumour ball forming process in Fig. 3 the application 3D culture medium
Seeding cells: inoculating cell；Medium change: replacement culture medium；
The tumour ball that the other cancerous cell lines of Fig. 4 are formed in the application 3D culture medium
The stemness related gene transcriptional level for the tumour ball that Fig. 5 the application 3D culture medium and conventional medium are formed compares
Wherein, ordinate indicates that mRNA expression multiple changes
The stemness correlative protein expression level for the tumour ball that Fig. 6 the application 3D culture medium and conventional medium are formed compares
From left to right, each swimming lane is respectively attached cell, the tumour ball of conventional medium formation, the application 3D culture medium shape
At tumour ball
Specific embodiment
It can be best understood from the present invention by the following examples, for illustration purposes, but be not limited to this hair
Bright claim.The present patent application institute is commercially available using reagent material.
Cancerous cell line used in this application: CNE cell line 5-8F, CNE cell line Sune1, National People's Congress's cell
Lung cancer cell line H460, human colon cancer cell line SW480.Above-mentioned cell line derives from triumphant base biology Co., Ltd.
Porcine HGF EGF, FGF and B27 used in this application are purchased from PeproTech company of the U.S..
1 3D of the embodiment culture selection of biomaterial
By a variety of biomaterial (methylcellulose (MC), gellan gum (GG), basement membrane matrixs, collagen, seaweed to be measured
Acid and agar) respectively with identical concentration, it is added to conventional medium (DMEM/F12+EGF 20ng/ml+FGF 20ng/ml+
B27 2%) in be configured to 3D culture medium, with 10000cells/ml human nasopharyngeal carcinoma 5-8F cell inoculation, observation is swollen after a week for culture
Tumor ball culture effect, MAIN OUTCOME MEASURES anti-adhesion ability, anti-settling ability, sample treatment difficulty and build cost.Synthesis is commented
Valence the results are shown in Table 1.
The feature of 1. various biological material of table.
0.3%MC is added in culture medium can effectively inhibit the fusion of 5-8F tumour ball, prevent as shown in Figure 1, cultivating containing MC
The tumour ball ratio of normal size is much larger than without MC culture medium in base.The fusion rate of 5-8F tumour ball is from 15.8 ± 3.9
Drop to 2.9 ± 1.0%, and similarly, the fusion rate of H460 tumour ball drops to 3.4 ± 0.7% from 19.7 ± 4.0, because
This MC can effectively inhibit the spontaneous fusion of tumour ball.
0.03%GG is added in culture medium can effectively prevent the sedimentation of tumour ball, as shown in Fig. 2, several in the culture medium without GG
It can't see suspension tumor ball, and tumour ball suspends in order in the culture medium containing GG, is distributed according to 3D.
Embodiment 2 prepares culture 3D culture systems culture medium
The scheme of 3D tumour ball culture systems: DMEM/F12+EGF 20ng/ml+FGF 20ng/ml+B272%+ methyl is fine
It ties up element (MC) 0.3%+ and ties cold foot (GG) 0.03%.
MC and GG saves the preparation of liquid: 3g MC and 0.3g GG powder is dissolved in respectively in the tri-distilled water of 100ml, under room temperature
It is sufficiently stirred 48 hours using magnetic stirring apparatus, dissolves fibre composition sufficiently；Mixing is placed in autoclave sterilizer,
160 DEG C, be greater than 2 hours, to remove endotoxin, temperature is down to room temperature after sterilizing, firmly shake it is repeated multiple times, up to no grumeleuse is
Only, set 4 DEG C it is spare, or packing after -20 DEG C preservation.
The preparation of culture medium in 3D culture systems: traditional tumour ball culture medium 500ml DMEM/F12+EGF is prepared first
20μg+FGF 10μg+B27 10ml；The prepared MC solution of 55ml and the configured GG solution of 5ml are added again；Final 3D is swollen
Tumor ball culture medium, which is prepared, to be completed.
Embodiment 3 largely prepares tumour ball using 3D culture medium.
Tumour ball incubation:
1. by the good purpose human nasopharyngeal carcinoma 5-8F cell dissociation of growth conditions and counting；
2. according to experiment purpose by certain amount (according to research purpose, 103-105Cells/ml cell inoculation) is trained to 3D
It supports in base, is cultivated in culture bottle or culture dish, 37 DEG C, 5%CO2。
3.3D culture systems change liquid: at the 3-5 days of tumour ball culture, carrying out culture medium replacement, be with solution turned yellow
Basic judgment criterion.
Change liquid process: 1) collect the 3D culture medium containing tumour ball into centrifuge tube, and be added isometric DMEM solution into
Row dilution；
2) centrifuge is centrifuged 200g, is centrifuged 10 minutes；
3) old culture medium is removed, new culture medium is added.
4. the formation that diameter is more than 100 μm of tumour ball can be observed after 5-7 days, isometric DMEM solution is added, from
The heart obtains tumour ball, carries out downstream research.
As shown in figure 3, at head two days of culture, due to non-adhering suspension culture environment, there are some natural death of cerebral cells, 5
After it, the partial size of tumour ball reaches 50-100 μm.After 7 days, tumour spherolite diameter reaches 100-200 μm, and the partial size of tumour ball
Uniform, the case where shape is round and smooth, fusion, is less.
And as shown in figure 4, Sune1, H460 and SW480 cell line can also form ideal tumour in the application 3D culture medium
Ball.
The Nature comparison for the tumour ball that the tumour ball and conventional medium that 4 the application 3D culture medium of embodiment is formed are formed
4.1CD133 detection
Method: the anti-CD133- phycoerythrin antibody of use (Miltenyi Biotec GmbH,
Bergisch Gladbach, Germany, catalog no.130-080-801,1:10) detection cell culture.
Attached cell and tumour ball are harvested, single cell suspension is decomposed by trypsase, and use CD133- in the dark at 4 DEG C
PE is dyed 30 minutes.Using flow cytomery staining cell, data analysis is carried out using FlowJo software 7.6.
5-8F cell and H460 cell are tested, the tumour ball institute that conventional medium and the application 3D culture medium obtain
The CD133 amount of expression is identical, is all 4 times of CD133 amount expressed by parent attached cell.
The detection of 4.2 stemness related genes
Method: attached cell is extracted using RNAiso Plus reagent (Takara Bio, Inc., Otsu, Japan) and is swollen
Tumor glomus cell total serum IgE.Pass through ultraviolet specrophotometer (ThermoNanoDrop 2000, Thermo Fisher
Scientific, Inc., Waltham, MA, USA) measurement RNA concentration.Then PrimeScript RT kit is used
(Takara Bio, Inc.), according to specification, carry out RT.Then, using SYBR Green RT-qPCR Master Mix
(Takara Bio, Inc.) carries out quantitative PCR.For eight aggressiveness combination transcription factors 4 (Oct-4), (sex-determining region Y)-box
The primer sequence of 2 (Sox2), Nanog and GAPDH are listed in tablei.GAPDH, which is used as, refers to gene.PCR reaction is related at 95 DEG C
Then lower denaturation 2 minutes keeps carrying out within 15 seconds 42 circulations at 95 DEG C, is kept for 30 seconds, then carried out most at 60 DEG C
Incubation afterwards continues 30 seconds at 72 DEG C.
Have detected 5-8F cell and H460 cell, as shown in figure 5, conventional medium and the application 3D culture medium formed it is swollen
The stemness related gene transcriptional level of tumor ball is approximate.
The detection of 4.30 stemness GAP-associated protein GAPs
Method: 5 minutes removing culture mediums, PBS washing are centrifuged with 200 × g to tumour ball and collected at room temperature in EP pipe
In.Next, tumour ball is fixed in EP pipe with 4% paraformaldehyde.Rabbit-anti mankind primary antibody, including Sox2 is added
(ab97959；1:1,000), Oct4 (ab19857；1:200) and Nanog (ab109250；1:200；It is all from Abcam), 4 DEG C
Concussion is incubated for overnight incubation.It is then cleaned with PBS and washes tumour ball three times, add the goat antirabbit secondary antibody (P0183 in conjunction with Cy3；
Beyotime Institute of Biotechnology；；1:1,500), tumour ball is incubated at room temperature 1 hour.DAPI
For nuclear staining (blue).Fluorescence microscope image.
Have detected 5-8F cell and H460 cell, as shown in fig. 6, conventional medium and the application 3D culture medium formed it is swollen
The stemness correlative protein expression of tumor ball is horizontal approximate.
Claims (6)
1. a kind of three-dimensional nodule ball culture medium additive, which is characterized in that contain methylcellulose and gellan gum.
2. culture medium additive according to claim 1, wherein the mass ratio of methylcellulose and gellan gum is 10:1.
3. a kind of three-dimensional nodule ball culture medium, ingredient is tumor cell culture culture medium, which is characterized in that also contains methyl
Cellulose and gellan gum.
4. culture medium according to claim 3, ingredient is DMEM/F12+EGF 20ng/ml+FGF 20ng/ml+B27
2%, the B27 are with volume ratio 1:50 addition, which is characterized in that also contain methylcellulose and gellan gum.
5. culture medium according to claim 3 or 4, wherein calculated by mass volume ratio, contain 0.3% Methyl cellulose
The plain gellan gum with 0.03%.
6. a kind of cultural method of three-dimensional nodule ball comprising following steps:
1) tumour cell is digested and is counted, and by the tumour cell digested by 103-105Cells/ml is inoculated into right and wants
It asks in the described in any item three-dimensional nodule ball culture mediums of 3-5,37 DEG C, 5%CO2Culture；
2) at the 3-5 days of tumour ball culture, until solution turned yellow, replaces culture medium；
The replacement culture medium operation are as follows:
The culture medium of the ball containing tumour is added into centrifuge tube, isometric DMEM solution dilution is added；
Centrifuge is centrifuged with 200g, is centrifuged 10 minutes；
Old culture medium is removed, new culture medium is added；
3) after cultivating 5-7 days, diameter to be formed is more than 100 μm of tumour ball, and isometric DMEM solution is added, and centrifugation obtains tumour
Ball.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110616185A (en) * | 2019-09-25 | 2019-12-27 | 华东理工大学青岛创新研究院 | Multi-cell tumor ball and high-flux preparation method thereof |
| CN112961821A (en) * | 2021-02-24 | 2021-06-15 | 四川大学华西医院 | Method for efficiently three-dimensionally culturing vascular endothelial cells |
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Application publication date: 20181214 |