CN110527666A - A kind of sub-sieve enrichment method of cancer stem cell - Google Patents
A kind of sub-sieve enrichment method of cancer stem cell Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0695—Stem cells; Progenitor cells; Precursor cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention provides a kind of sub-sieve enrichment method of cancer stem cell, comprising steps of preparing ultralow absorbing agar gel entrapment culture ware or porous plate；Prepare cancer stem cell complete medium；The single celled suspension of cancer cell is prepared, and washes away residual serum；Under serum-free condition, unicellular by cancer cell is inoculated in low adsorption agar gel culture dish or porous plate, and the cancer stem cell complete medium is added, unicellular, the acquisition cancer stem cell microsphere of cancer cell described in suspension balling-up culture；The used low adsorption agar gel culture dish or porous plate are cleaned, then carries out the cancer stem cell microsphere secondary culture in low adsorption agar plates or porous plate after cleaning.Equipment and operation are required it is simple and convenient, save the time, cost it is very low and can safety reuse；It can be enriched with to obtain the cancer stem cell of a large amount of high quality suitable for various types of cancers；And experiment repetitive rate of the invention has higher guarantee.
Description
Technical field
The present invention relates to technical field of cell culture more particularly to a kind of sub-sieve enrichment methods of cancer stem cell.
Background technique
Cancer is a kind of different substantiality disease, is made of many different types of cancer cells.More and more researches show that
There is a small set of cells to have a variety of features similar with normal stem cell, including self-renewal capacity and multidirectional point in cancer
Change potential, referred to as cancer stem cell (cancer stem cells, CSCs) or tumour initiator cell (tumor
initiating cells).A large amount of evidence confirmation cancer stem cell is in the starting and relapsing course of cancer, the production of drug resistance
It plays an important role in terms of raw and transfer stove formation.Conventional radiotheraphy or chemotherapy for cancer can kill major part
Tumour cell, but cancer stem cell can be survived and be generated by self-renewal capacity and multi-lineage potential new
Complete heterogeneous.Subsequent therapeutic scheme is unsatisfactory to the therapeutic effect of these late malignant tumours.Therefore, needle
There is particularly important meaning to cancer is captured to the screening enrichment and the research of effective treatment method of cancer stem cell.
So far, researcher is dry thin from cancer is isolated derived from a variety of entity tumors or its in cell line
Born of the same parents, including leukaemia, prostate cancer, the cancer of the brain, breast cancer, colon cancer, lung cancer and cancer of pancreas etc..But since cancer is dry thin
Born of the same parents' ratio shared in tumour is very rare, must just separate cancer stem cell and expand culture in order to carry out
Research.Method used in document mainly has side population cell sub-sieve method, Specific marker sub-sieve method and suspended microspheres at present
Body cultivation.
Side population cell (side population cells) sub-sieve method is the powerful outlet ability based on cancer stem cell
(higher abc transport protein expression: such as ABCG2, MDR1 or MRP1) makes dyestuff (Hoechst 33342) discharge cell
Cancer stem cell cannot be coloured, so as to be come out its sub-sieve by flow cytometry.The jack to jack adapter obtained by the method
Property side population cell is proved to stem cell properties and has stronger tumorigenesis ability and drug resistance.The side group that sub-sieve obtains
Cell is not stem cell entirely, and it is heterogeneous or stronger.
Specific marker sub-sieve method is the albumen specific expressed using cancer stem cell, with fluidic cell fluorescence sub-sieve
Technology (fluorescence activated cell sorting, FACS) or immunological magnetic bead sorting method (magnetic
Activated cell sorting, MACS) sub-elect cancer stem cell.Currently used Specific marker have CD44,
CD133, CD24, ALDH1A1, ABCG2, BMI1 etc..Since the Specific marker that different cancers is expressed is not fully identical,
So being applied to all cancers there is no pervasive cancer stem cell Specific marker carrys out sub-sieve cancer stem cell.
Above method needs to have higher requirements to equipment and operation using flow cytometry, and cell is by instrument
Reason, can generate some unknowable influences, therefore the culture in its later period and the operation of experiment can be held to the characteristic of cancer stem cell
Continuous property and repeatability are unsatisfactory.Further, since the rare characteristic of cancer stem cell, these methods are also not suitable for largely being enriched with
Cancer stem cell.
Microactuator suspension sphere (spheroid or sphere) cultivation be by cancer cell in the form of unicellular in serum-free item
It is incubated under part in the incubator of ultralow absorption surface, the characteristic of microsphere can be grown up to using only stem cell come sub-sieve
It is enriched with cancer stem cell.There is no an adherent contact stimulus, ordinary cells will stop growing and gradually apoptosis, and cancer stem cell
But it can survive and be proliferated.Removing serum is in order to break up cancer stem cell not, because of some factor energy in serum
Enough stimulation cancer stem cells break up.Each microsphere that the method obtains be from the growth of cancer stem cell,
It is especially advantageous for studying cancer stem cell on individual cell level.Therefore, it can be obtained by continuous serum free suspension secondary culture
To the very high cancer stem cell of purity, and requirement of the method to equipment and operation is very low, very useful rich in a large amount of sub-sieves
Collect cancer stem cell.At present there are many ultralow absorption culture dishes used in document, such as with the coated culture dish of PolyHEMA.
These method kinds have more expensive, some then more complicated time-consumings.
Lack a kind of sub-sieve enrichment method of simple, reliable, economic, efficient cancer stem cell in the prior art.
Summary of the invention
The present invention in order to solve the problems in the prior art, provides a kind of sub-sieve enrichment method of cancer stem cell.
To solve the above-mentioned problems, the technical solution adopted by the present invention is as described below:
A kind of sub-sieve enrichment method of cancer stem cell, includes the following steps: S1: preparing ultralow absorbing agar gel entrapment culture
Ware or porous plate；S2: cancer stem cell complete medium is prepared；S3: the single celled suspension of cancer cell is prepared, and is washed away residual
Stay serum；S4: under serum-free condition, by the cancer cell it is unicellular be inoculated in the low adsorption agar gel culture dish or
In porous plate, and the cancer stem cell complete medium is added, unicellular, the acquisition of cancer cell described in suspension balling-up culture
Cancer stem cell microsphere；S5: the used low adsorption agar gel culture dish of cleaning or porous plate, then after cleaning
The cancer stem cell microsphere secondary culture is carried out in the low adsorption agar plates or porous plate.
Preferably, step S1 includes the following steps: S11: the agar solution of 1.2%-4.0%, high temperature are prepared with ultrapure water
The liquid condition of agar solution is maintained after high pressure sterilization in 40 DEG C of -50 DEG C of water-baths；S12: by the agar solution and 2 ×
DMEM/F12 culture medium 1:1 is laid in the bottom of culture dish or porous plate at once after mixing, stands 15-30 minutes to agar
Gel solidifies completely, obtains the ultralow absorbing agar gel entrapment culture ware or porous that agar gel concentration is 0.6%-2.0%
Plate.
Preferably, step S2 includes the following steps: the people that final concentration of 15-25ng/ml is added into the DMEM/F12
Recombinant human epidermal growth factor, people's recombination basic fibroblast growth factor of final concentration of 10-25ng/ml, final concentration of 3-8
The insulin of μ g/ml, it is final concentration of 1 × B-27 additive, final volume is than the serum substitute, final concentration of for 1%-15%
Blueness-streptomysin of 100U/ml is uniformly mixed and obtains the cancer stem cell complete medium.
Preferably, step S3 includes the following steps: to be digested to unicellular with pancreatin by cancerous tissue or culture and uses phosphorus
Phthalate buffer washes away remaining serum.
Preferably, it is 37 DEG C, CO that culture described in step S4, which is in temperature,2It is cultivated in the incubator that concentration is 5%.
Preferably, step S5 include: collect the cancer stem cell microsphere with settling methods or strainer filtering method, and
The low adsorption agar plates or porous plate are cleaned with phosphate buffer, the cancer stem cell microsphere pancreatin is disappeared
Chemical conversion microsphere is unicellular and counts, by low adsorption agar plates or porous plate described in the unicellular renewed vaccination of the microsphere
Carry out microsphere culture.
Preferably, the concentration of agar gel is done by the cancer in the ultralow absorbing agar gel entrapment culture ware or porous plate
The expression of the numbers of cell microspheres, size and cancer stem cell Specific marker determines.
Preferably, it is thin to be recycled and reused for same cancer for the used ultralow absorbing agar gel entrapment culture ware or porous plate
The suspension culture of born of the same parents.
Preferably, it is inoculated in after the low adsorption agar gel culture dish or porous plate originally in step S4 by unicellular
In 4 days, the ultralow absorbing agar gel entrapment culture ware or porous plate cannot have any movement.
Preferably, be inoculated in the ultralow absorbing agar gel entrapment culture ware of 10cm the cancer cell number be 1 ×
104-1×105It is a；The every hole inoculating cell number of the ultralow absorbing agar gel entrapment culture porous plate in 6 holes is 500-5000.
The invention has the benefit that provide a kind of sub-sieve enrichment method of cancer stem cell, method of the invention is to setting
Standby and operation requires simple and convenient, saves the time, cost it is very low and can safety reuse；Suitable for various types of
Cancer can be enriched with to obtain the cancer stem cell of a large amount of high quality；And experiment repetitive rate of the invention has higher guarantee, can
A large amount of reliable cancer stem cells are provided with the research work for vast cancer stem cell, have promotion to anticipate cancer clinical treatment
Justice.
Detailed description of the invention
Fig. 1 is the sub-sieve enrichment method schematic diagram of cancer stem cell in the embodiment of the present invention 1.
Fig. 2 is the method schematic diagram that ultralow absorbing agar gel entrapment culture ware or porous plate are prepared in the embodiment of the present invention 1.
Fig. 3 (a) be in the embodiment of the present invention 2 DU145 cell agar concentration be 0.6% when agar gel on grow it is micro-
The aspect graph of sphere.
Fig. 3 (b) be in the embodiment of the present invention 2 DU145 cell agar concentration be 0.9% when agar gel on grow it is micro-
The aspect graph of sphere.
Fig. 3 (c) be in the embodiment of the present invention 2 DU145 cell agar concentration be 1.2% when agar gel on grow it is micro-
The aspect graph of sphere.
Fig. 3 (d) be in the embodiment of the present invention 2 DU145 cell agar concentration be 1.5% when agar gel on grow it is micro-
The aspect graph of sphere.
Fig. 3 (e) be in the embodiment of the present invention 2 DU145 cell agar concentration be 2.0% when agar gel on grow it is micro-
The aspect graph of sphere.
Fig. 4 is the number statistical figure for counting microsphere in the embodiment of the present invention 2 under different agar concentrations.
Fig. 5 is the statistical chart for counting the diameter of microsphere in the embodiment of the present invention 2 under different agar concentrations.
Fig. 6 is the qRT-PCR analysis result of the cancer stem cell Specific marker of prostate cancer in the embodiment of the present invention 2
Schematic diagram.
Fig. 7 is that the facs analysis result of the cancer stem cell Specific marker of prostate cancer in the embodiment of the present invention 2 is shown
It is intended to.
Fig. 8 (a) be in the embodiment of the present invention 2 prostate gland cancer cell cell number be 1 × 103When attached cell (Ad) and
Growth tumorigenesis situation schematic diagram of the microsphere (Sp) in SCID mice back.
Fig. 8 (b) be in the embodiment of the present invention 2 prostate gland cancer cell cell number be 1 × 104When attached cell (left back)
And microsphere (right back) is in the growth tumorigenesis situation schematic diagram of SCID mice back.
Fig. 9 (a) is the aspect graph of attached cell in the embodiment of the present invention 2.
Fig. 9 (b) is the aspect graph of first generation microsphere in the embodiment of the present invention 2.
Fig. 9 (c) is the aspect graph of second generation microsphere in the embodiment of the present invention 2.
Fig. 9 (d) is the aspect graph of third generation microsphere in the embodiment of the present invention 2.
Fig. 9 (e) is the aspect graph of attached cell again in the embodiment of the present invention 2.
Figure 10 is the result schematic diagram of continuous passage 3 times in the embodiment of the present invention 2 DU145 microsphere number statisticals.
Figure 11 is the microsphere and microsphere of the DU145 of adherent growth in the embodiment of the present invention 2, the first generation to the third generation
The analysis comparing result schematic diagram of the cancer stem cell Specific marker of the DU145 of adherent growth again.
Figure 12 be the cancer cell of various cancers type in the embodiment of the present invention 3 suspend on agar gel culture dish culture life
The form schematic diagram of long attached cell and microsphere.
Figure 13 is the cancer stem cell Specific marker of prostate cancer LNCaP microsphere in the embodiment of the present invention 3
QRT-PCR analyzes result schematic diagram.
Figure 14 is the cancer stem cell Specific marker of prostate cancer LNCaP microsphere in the embodiment of the present invention 3
Facs analysis result schematic diagram.
Figure 15 is the carcinoma of prostate stem cell specific negative of prostate cancer LNCaP microsphere in the embodiment of the present invention 3
The expression schematic diagram of marker.
Figure 16 is the carcinoma of prostate stem cell specific negative mark of prostate cancer VCaP microsphere in the embodiment of the present invention 3
The expression schematic diagram of will object.
Specific embodiment
The present invention is described in detail by specific embodiment with reference to the accompanying drawing, for a better understanding of this hair
It is bright, but following embodiments are not intended to limit the scope of the invention.In addition, it is necessary to illustrate, diagram provided in following embodiments
The basic conception that only the invention is illustrated in a schematic way, in attached drawing only display with related component in the present invention rather than according to reality
Component count, shape when implementation and size are drawn, when actual implementation each component shape, quantity and ratio can for it is a kind of with
The change of meaning, and its assembly layout form may also be increasingly complex.
Embodiment 1
As shown in Figure 1, the present invention provides a kind of sub-sieve enrichment method of cancer stem cell, Integral Thought is first by cancer
Cell dissociation is resuspended in serum free medium after washing away residual serum, is then inoculated in ultralow suction of the invention at unicellular
Culture growth microsphere in attached culture dish/porous plate.
Specifically comprise the following steps:
1. preparing ultralow absorbing agar gel entrapment culture ware or porous plate；
As shown in Fig. 2, specifically comprising the following steps:
(1) agar solution that 1.2%-4.0% is prepared with ultrapure water, in 40 DEG C of -50 DEG C of water-baths after autoclave sterilization
Maintain the liquid condition of agar；
(2) agar and 2 × DMEM/F12 culture medium 1:1 are laid in culture dish or porous plate at once after mixing
Bottom, stand and solidified completely to agar gel for 15-30 minute, obtaining agar gel concentration is that the described of 0.6%-2.0% surpasses
Low adsorption agar gel culture dish or porous plate.
In a kind of specific embodiment, the preparation (sterile working) of ultralow absorbing agar culture dish or 6 orifice plates includes:
(1) agar is dissolved in ultrapure water, is made into 1.2%, 1.8%, 2.4%, 3.0% and 4.0% (weight g: volume ml)
Concentration, autoclave sterilization is cooled to 50 DEG C or so and is put into the liquid conditions that 42 DEG C of water-baths maintain agar；
(2) 2 × DMEM/F12 culture medium is put into 42 DEG C of water-bath warm bath；
It can be understood that the temperature of water-bath may be 40 DEG C or 50 DEG C in above-mentioned steps, feelings according to specific experiments
Condition is selected.
(3) in superclean bench, according to the volume ratio of 1:1, agar solution is mixed in 2 × DMEM/F12 culture medium
In, it is uniformly mixed.It is poured into 10cm culture dish or 6 orifice plates at once, and rotates 10cm culture dish or 6 orifice plates make agar-
DMEM/F12 mixed liquor uniformly covers entire bottom surface.10cm culture dish needs the agar-DMEM/F12 of 8-15ml to mix
Liquid, 6 orifice plates need the agar-DMEM/F12 mixed liquor of 1-2ml.
(4) 10cm culture dish or 6 orifice plates 15-30 minutes are stood, are fully cured to agar gel.The final concentration of agar is distinguished
It is 0.6%, 0.9%, 1.2%, 1.5%, 2.0%.The agar 10cm culture dish or 6 orifice plates made can be used directly,
It is sealed after suitable cancer stem cell complete medium can be added, stores 1 week or 4 DEG C of refrigerator storages 2 in 37 DEG C of incubators
Week.
2. preparing cancer stem cell complete medium；
In superclean bench, it is (dense eventually that rhEGF is added into DMEM/F12GlutaMAX culture medium
Spend 15-25ng/ml), people's recombination basic fibroblast growth factor (final concentration 10-25ng/ml), insulin (final concentration 3-8
μ g/ml), B-27 additive (final concentration 1 ×), KnockOut serum substitute (final volume ratio 1%-15%), blueness-streptomysin
(final concentration 100U/ml) is uniformly mixed.It can be stored 2-4 weeks in 4 DEG C of refrigerators.
3. preparing the single celled suspension of cancer cell, and wash away residual serum；
It may include steps of in a kind of specific embodiment:
(1) with pancreatin (TrypLE) various types of cancerous tissues or culture are digested to unicellular and are resuspended in phosphoric acid
It in salt buffer, is collected by centrifugation, then is washed 1-2 times with phosphate buffer, to remove remaining serum；It is dry to be finally resuspended in cancer
In cell culture complete medium；
(2) under the microscope, it observes single celled cell number and calculates cell concentration；
(3) the agar 10cm culture dish prepared or 6 orifice plates are taken out, suitable cancer cell is inoculated with and supplements fresh cancer
Stem cell complete medium.For 10cm culture dish, the optimum cell number of inoculation is 1 × 104-1×105A cancer cell, needs
The culture medium of 7-10ml；The best inoculating cell number of 6 orifice plates is 500-5000 cancer cell, needs 1.5-2ml culture medium；
(4) putting inoculated agar plates or 6 orifice plates into temperature is 37 DEG C, CO2The incubator that concentration is 5%, and
Culture dish or 6 orifice plates cannot have any movement in initial 4 days.The fresh cancer stem cell of supplement 1-2ml is complete within every 3-4 days
Culture medium；
(5) cultivate 1-4 week, collect diameter be greater than 50 μm microsphere (gravitational settling 3-5 minutes or 50-100 μm of aperture
Sterile strainer filtering), carry out follow-up study experiment.These microspheres are grown from a cancer stem cell, the inside packet
The cancer stem cell of high quality is contained；
(6) microsphere of cancer stem cell is passed on.It operates and passes on according to step (1)-(4) after collection.With passage time
Number increases, and the ratio regular meeting of cancer stem cell sharply increases, and the cell number of inoculation can be reduced suitably.It passage 6 times or more, can get
The cancer stem cell of higher degree.
In alternative embodiments of the invention, the selection of agar gel optimal concentration.Agar concentration is bigger, and hardness is got over
Greatly, the contact of cell is supported also just smaller.For different cancer cells, while being inoculated in the agar 6 orifice plates of multiple concentration
(0.6%-4.0%), the expression according to the number of growth microsphere, size and cancer stem cell Specific marker are true
Determine optimal concentration.
In another alternative embodiments of the invention, the reuse of agar plates.In microsphere of the same race passage,
The remaining cell of used agar plates is washed away with phosphate buffer, can continue to be inoculated with passage cell.One block of fine jade
Rouge culture dish can be continually used for the secondary culture of microsphere of the same race.Wherein the various nutrition of cancer stem cell complete medium because
Into agar gel, fully penetrated balance completely, is very suitable to continue to cultivate microsphere amplification cancer stem cell.
In another alternative embodiments of the invention, step 1. prepares ultralow absorbing agar gel entrapment culture ware or porous
Plate, 2. prepare the single celled suspension of cancer stem cell complete medium and 3. preparation cancer cells, and wash away residual serum
Can synchronize and carry out any two of them step or three steps, can also according to 3-2-1 or 2-1-3 or 3-1-2 or 2-3-1 or
The step of 1-3-2, carries out.
Embodiment 2
It is enriched with using the sub-sieve of method of the invention to the cancer stem cell of human prostata cancer DU145 cell line, wherein institute
The primer used is as shown in table 1.
1 list of primers of table
Specifically comprise the following steps:
1.DU145 cell line is bought in ATCC.Condition of culture are as follows: DMEM culture medium supplements 10% fetal calf serum and blueness-
Streptomysin, in 37 DEG C, 5%CO2It is cultivated in incubator.
2. the agar solution of 1.2%, 1.8%, 2.4%, 3.0% and 4.0% (weight g: volume ml) is configured with ultrapure water,
Autoclave sterilization is cooled to 50 DEG C or so and is put into the liquid condition that 42 DEG C of water-baths maintain agar.Take out suitable 2 ×
DMEM/F12 culture is based on 42 DEG C of water-bath heating.
3. rapid operation, the 2 × DMEM/F12 culture for drawing 4ml preheating is based in 15ml centrifuge tube, then draws 4ml heat
1.2% agar solution is mixed in 15ml centrifuge tube, is mixed, and is drawn 1ml at once in 1 hole of 6 orifice plates, is repeated 3 holes,
Rocking makes agar solution that entire bottom surface be completely covered.It is statically placed in ultra-clean station 10 minutes, gently picks up orifice plate inspection
Whether solidify completely.This is 0.6% agar gel culture plate.
4. distinguishing the agar gel culture plate of paved 0.9%, 1.2%, 1.5 and 2.0% according to step 3.
5. preparing cancer stem cell complete medium.Epidermal growth factor is added into DMEM/F12GlutaMAX culture medium
EGF (final concentration 20ng/ml), basic fibroblast growth factor bFGF (final concentration 20ng/ml), insulin (5 μ of final concentration
G/ml), B-27 additive (final concentration 1 ×), KnockOut serum substitute (final concentration 1%), blueness-streptomysin (final concentration
100U/ml), it is uniformly mixed.It can be stored 1 month in 4 DEG C of refrigerators.It is heated using preposition in 37 DEG C of water-baths.
6. the cancer stem cell complete medium of 1.5ml is added into the agar plate prepared in step 3, it is put into 37
DEG C incubator is spare.
7. digesting the cell of DU145 adherent growth in 37 DEG C using pancreatin, (disappeared according to different cell line optimization within 3 minutes
Change the time), resuspension is gently blown and beaten with the culture medium containing serum, 200g is centrifuged 3 minutes collection cells；Phosphate buffer cleaning 2
It is secondary, it is resuspended in cancer stem cell complete medium.
8. being counted under microscope.Agar gel 6 orifice plates are taken out, and are inoculated with 1500 DU145 cells of equivalent to each hole.
9. putting back to 37 DEG C, 5%CO2It is cultivated in incubator.Originally irremovable culture plate in 4 days.Since the 5th day,
Interval supplements the cancer stem cell complete medium 0.5ml of fresh preheating for 3-4 days into agar plate.
In the growing state of microscopically observation monitoring microsphere.Fig. 3 (a)-Fig. 3 (e) is DU145 cell in agar concentration
The aspect graph of the microsphere grown on agar gel when being 0.6%, 0.9%, 1.2%, 1.5%, 2.0%.Each microsphere
It is being grown by a cell by self-renewal capacity, so the quantity of microsphere can be used to measure the 1500 of inoculation
The quantity of cancer stem cell in a cancer cell.Document, which is summarized, thinks that microsphere of the diameter greater than 50 μm is just considered cancer
Stem cell growth and come microsphere.
As shown in Figure 4 and Figure 5, the number of microsphere is counted under different agar concentrations and measures its diameter.Knot
Fruit show microsphere quantity in 0.9% agar plate at most and with the concentration of agar gel increase, number by
It is decrescence few.Equally, the diameter of microsphere increases also with the concentration of agar gel and is reduced.Therefore, 0.9% agar is solidifying
Glue is chosen to do next research experiment.
10. the agar plates of preparation 0.9%.According to step 2-3, by 1.8% agar solution of 5ml be mixed into 5ml 2 ×
In DMEM/F12 culture medium, mixes and poured into 10cm culture dish at once, rocking makes agar solution that entire bottom table be completely covered
Face is statically placed in ultra-clean station 15-30 minutes.It gently picks up culture dish and checks whether complete solidification, 7ml cancer is then added
It is spare to be put into 37 DEG C of incubators for stem cell complete medium.
11. preparing the single cell suspension of DU145 in cancer stem cell complete medium, count, inoculation 5 × 104It is a thin
Born of the same parents rock mixing in 0.9% agar plates.
12. putting back to 37 DEG C, 5%CO2It is cultivated in incubator.Originally irremovable culture plate in 4 days.Since the 5th day,
Interval supplements the cancer stem cell complete medium 1-3ml of fresh preheating for 3-4 days into agar plate.
It collects first generation microsphere P1 (gravitational settling 3 minutes or sterile strainer filtering), is digested to unicellular outstanding after 2-3 weeks
Liquid carries out following experiment:
(1) it extracts RNA and carries out qRT-PCR experiment, verify the expression of cancer stem cell Specific marker.Prostate cancer is thin
The stem cell properties for the microsphere that born of the same parents (DU145) grow in 0.9% agar plates are analyzed as a result, result such as Fig. 6 and Fig. 7 institute
Show.Fig. 6 is the qRT-PCR analysis result schematic diagram of the cancer stem cell Specific marker of prostate cancer, wherein hollow cylindrical
Attached cell is represented, solid cylindricality represents microballoon body cell；Fig. 7 is the cancer stem cell Specific marker of prostate cancer
The facs analysis result schematic diagram of (CD44 and CD133).Kinds cancer stem cell Specific marker, such as CD133, CD44,
The expression quantity in microsphere such as NANOG, BMI1, which has, significantly to be increased.Moreover, Fig. 7 shows CD44+/CD133+ in microsphere
Double positive cells improve also significantly.
(2) it is inoculated in the back of SCID mice, verifies its tumorigenesis ability.As a result such as Fig. 8 (a) and Fig. 8 (b) display, Fig. 8
It (a) be prostate gland cancer cell in cell number is 1 × 103When attached cell and microsphere SCID mice back growth tumorigenesis
Situation；It is 1 × 10 that Fig. 8 (b), which is prostate gland cancer cell in cell number,4When attached cell and microsphere in SCID mice back
Grow tumorigenesis situation；A left side is measured as attached cell in two figures, and right back side is microballoon body cell, as we can see from the figure microsphere
That is the mouse tumorigenesis ability of cancer stem cell is significantly stronger than the cancer cell of adherent growth.
Further experimental result is as shown in table 2.
The growth tumorigenesis situation of the attached cell of 2 prostate gland cancer cell of table and microsphere in SCID mice back
(3) 1000 cell inoculations taking out in 0.9% agar 6 orifice plates, this is second generation microsphere P2 for culture 1-3 week,
Count microsphere number.
13. carrying out microsphere secondary culture in the same agar plates.Agar culture is cleaned with phosphate buffer
To wash away residual cell, 1 × 10 is taken ware 2 times4A microsphere P1 cell renewed vaccination is cultivated 1-2 weeks.
14. by continuous passage 3 times DU145 microsphere cell inoculations in 60mm routine culture ware, routinely cultural method
Culture, this microsphere adherent DU145 again.By continuous passage 3 times DU145 microspheres, microsphere again adherent DU145 cell with
DU145 attached cell compares and analyzes, and is the adherent growth of DU145 cell line as shown in Fig. 9 (a) -9 (e), same
Continuous passage culture microsphere and aspect graph in 0.9% agar plates；If Figure 10 is continuous passage 3 times DU145 microballoons
The result schematic diagram of body number statistical；Figure 11 is the microsphere and microsphere of the DU145 of adherent growth, the first generation to the third generation
The analysis comparing result schematic diagram of the cancer stem cell Specific marker of the DU145 of adherent growth again.
As the result is shown with the increase of passage number, in the cell of culture that suspends on agar gel shared by cancer stem cell
Ratio it is higher and higher.100% is not achieved for ratio shared by cancer stem cell, document is construed in microsphere, with
To going deep into inside microsphere, nutrition and oxygen can be fewer and fewer, and which results in the cells of microsphere core, and apoptosis has occurred,
To which the one layer of cells of only microsphere surface contains largely active cancer stem cell.
Embodiment 3
It is enriched with using the sub-sieve of method of the invention to a variety of different types of cancer stem cells, specifically includes following step
It is rapid:
1. human prostate cancer cell line LNCaP, VCaP, human colon cancer cell line HCT116 and hepatocellular carcinoma cells system
Hep G2 is bought in ATCC.LNCaP is incubated at 1640 culture medium of RPMI, VCaP is incubated at DMEM culture medium, HCT116 culture
It is incubated at McCoy's 5a culture medium in MEM culture medium, Hep G2, supplements 10% fetal calf serum and blueness-streptomysin.
2. the agar gel culture dish of preparation 0.9% prepares cancer stem cell complete medium.
3. by cancer cell in 37 DEG C, 5%CO2Adhere-wall culture is digested to single cell suspension, washes away residual to 60-80% density
Serum is stayed, cancer stem cell complete medium is resuspended in.
4. counting, inoculation 1 × 105A cancer cell is in the agar culture containing 7ml cancer stem cell complete medium 0.9%
In ware.
5.37 DEG C are cultivated 2-4 weeks.The growing state of microscopically observation microsphere is as shown in figure 12 various cancers class
The cancer cell of type suspends on agar gel culture dish cultivates attached cell and the form schematic diagram of microsphere of growth.
6. extracting RNA for the microsphere of LNCaP and VCaP, after collection carries out qRT-PCR analysis, as a result such as Figure 13-figure
16 displays, microsphere express higher cancer stem cell Specific marker NANOG, CD133 etc. and prostate cancer cancer
Stem cell specificity low expression AR, PSA etc..
The foregoing describe basic principles and main features of the invention, those skilled in the art is it should be appreciated that the present invention
It is not limited to the above case study on implementation, the sub-sieve that can be applicable to other cancer stem cells is enriched with and makees adjustment appropriate.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those skilled in the art to which the present invention belongs, it is not taking off
Under the premise of from present inventive concept, several equivalent substitute or obvious modifications can also be made, and performance or use is identical, all answered
When being considered as belonging to protection scope of the present invention.
Claims (10)
1. a kind of sub-sieve enrichment method of cancer stem cell, which comprises the steps of:
S1: ultralow absorbing agar gel entrapment culture ware or porous plate are prepared；
S2: cancer stem cell complete medium is prepared；
S3: the single celled suspension of cancer cell is prepared, and washes away residual serum；
S4: under serum-free condition, by the unicellular low adsorption agar gel culture dish or more of being inoculated in of the cancer cell
In orifice plate, and the cancer stem cell complete medium is added, unicellular, the acquisition cancer of cancer cell described in suspension balling-up culture
Disease stem cell microsphere；
S5: the used low adsorption agar gel culture dish of cleaning or porous plate, then the low adsorption fine jade after cleaning
The cancer stem cell microsphere secondary culture is carried out in rouge culture dish or porous plate.
2. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that step S1 includes following step
It is rapid:
S11: the agar solution of 1.2%-4.0% is prepared with ultrapure water, is maintained after autoclave sterilization in 40 DEG C of -50 DEG C of water-baths
The liquid condition of agar solution；
S12: the agar solution and 2 × DMEM/F12 culture medium 1:1 are laid in culture dish or porous at once after mixing
The bottom of plate stands 15-30 minutes and solidifies completely to agar gel, and obtaining agar gel concentration is the described of 0.6%-2.0%
Ultralow absorbing agar gel entrapment culture ware or porous plate.
3. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that step S2 includes following step
It is rapid: rhEGF, the final concentration of 10- of final concentration of 15-25ng/ml being added in Xiang Suoshu DMEM/F12
People's recombination basic fibroblast growth factor of 25ng/ml, final concentration of 3-8 μ g/ml insulin, it is final concentration of 1 ×
B-27 additive, final volume are uniformly mixed than blueness-streptomysin of serum substitute, final concentration of 100U/ml for 1%-15%
Obtain the cancer stem cell complete medium.
4. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that step S3 includes following step
It is rapid: cancerous tissue or culture being digested to unicellular with pancreatin and wash away remaining serum with phosphate buffer.
5. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that cultivating described in step S4 is
It is 37 DEG C, CO in temperature2It is cultivated in the incubator that concentration is 5%.
6. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that step S5 includes: to use gravity
Sedimentation or strainer filtering method collect the cancer stem cell microsphere, and clean the low adsorption agar with phosphate buffer
It is unicellular and count with pancreatin to be digested to microsphere by culture dish or porous plate for the cancer stem cell microsphere, will be described micro-
Low adsorption agar plates described in the unicellular renewed vaccination of sphere or porous plate carry out microsphere culture.
7. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that the ultralow absorbing agar is solidifying
In glue culture dish or porous plate the concentration of agar gel by the cancer stem cell microsphere number, size and cancer stem cell
The expression of Specific marker determines.
8. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that the used ultralow suction
Attached agar gel culture dish or porous plate are recycled and reused for the suspension culture of same cancer cell.
9. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that connect in step S4 by unicellular
It plants in 4 days after the low adsorption agar gel culture dish or porous plate originally, the ultralow absorbing agar gel entrapment culture ware
Or porous plate cannot have any movement.
10. the sub-sieve enrichment method of cancer stem cell as described in claim 1, which is characterized in that the ultralow suction of 10cm
The number that the cancer cell is inoculated in attached agar gel culture dish is 1 × 104-1×105It is a；The ultralow absorbing agar gel in 6 holes
Cultivating the every hole inoculating cell number of porous plate is 500-5000.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254116A (en) * | 2020-01-18 | 2020-06-09 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Method for enriching and flow-sorting human ovarian cancer stem cells |
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