CN108085296A - Culture medium and application thereof - Google Patents
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- CN108085296A CN108085296A CN201810083614.9A CN201810083614A CN108085296A CN 108085296 A CN108085296 A CN 108085296A CN 201810083614 A CN201810083614 A CN 201810083614A CN 108085296 A CN108085296 A CN 108085296A
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Abstract
The present invention proposes a kind of culture medium.The culture medium includes：Basal medium, serum substitute, dual anti-, glutamate/ester, N acetylcysteines, CHIR 99021 and LDN 193189.Using culture medium in vitro culture intestinal crypts according to embodiments of the present invention or intestinal stem cell, the quantity of the intestinal crypts of acquisition is more than the prior art, the ratio higher of the stem cell in the intestinal crypts of acquisition, the intestinal stem cell of acquisition it is more；And using the dryness that according to the culture medium in vitro culture intestinal crypts of this year inventive embodiments, can maintain the stem cell in intestinal crypts for a long time, cell Proliferation is more vigorous in intestinal crypts, and cell differentiation is normally orderly in intestinal crypts.Culture medium according to embodiments of the present invention is beneficial to the popularization of gut epithelial stem cells culture systems and intestinal crypts organoid culture systems, is conducive to intestinal stem cell and largely expands in vitro, and then provides very favorable platform for the treatment of intestines problem.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to culture medium and application thereof, more specifically, this hair
The bright purposes for being related to culture medium, CHIR-99021 and LDN-193189 in culture medium is prepared, cultivate intestinal crypts method with
And the method for obtaining intestinal stem cell.
Background technology
Enteric epithelium is the primary structure that human body performs digestion and absorption function, its update is very rapid, and its update is main
It is to be driven by being located at the gut epithelial stem cells in its bottom crypts structure, thus the research to intestinal stem cell and health
It is closely bound up.In recent years, the foundation of gut epithelial stem cells vitro culture system has promoted the research to gut epithelial stem cells significantly,
Convenient for directly being operated and being observed to gut epithelial stem cells in vitro.The gut epithelial stem cells of these in vitro cultures can be with whole
It closes into the enteric epithelium after damage, helps to repair the damage of enteric epithelium, be hopeful to be applied to the treatment of intestinal tract injury disease.
When carrying out Murine intestinal epithelium organoid culture, the condition of culture used is：Basal medium Advanced
DMEM/F12, serum substitute N2 and B27, the N-acetylcysteine of dual anti-, 2mM GlutaMAX and 1mM, and it is required
The growth factor wanted：The R-spondin 1 of the Noggin and 1ug/mL of EGF, 100ng/mL of 50ng/mL.It is unicellular in progress
, it is necessary to add in the Wnt-3a of the Blebbistatin and 100ng/mL of 10uM on the basis of organoid culture medium during culture.Into
, it is necessary to add in the Wnt-3a of 100ng/mL on the basis of small intestine culture medium during row mouse Colon organoid in vitro culture.It carries out
The Wnt-3a of the addition 100ng/mL on the basis of mouse small intestine crypts culture medium is needed during the small intestine culture of people, 500nM's
[the Leu15]-Gastrin I of the Nicotinamide and 100uM of the SB202190 of A83-01,10uM, 1M.
Existing intestinal stem cell vitro culture system need add in EGF, Noggin and R-spondin (ENR) or ENR and
The growth factors such as Wnt-3a (ENRW), these growth factors are expensive, are unfavorable for the popularization of gut epithelial stem cells culture systems,
It especially limits intestinal stem cell largely to expand in vitro, for the treatment of intestines problem.
The content of the invention
It is contemplated that it solves at least some of the technical problems in related technologies.
For this purpose, in the first aspect of the present invention, the present invention proposes a kind of culture medium.According to an embodiment of the invention, institute
Stating culture medium includes：Basal medium, serum substitute, dual anti-, glutamate/ester, N-acetylcystein, CHIR-99021
And LDN-193189.Culture medium according to embodiments of the present invention using CHIR-99021 and LDN-193189 substitute EGF,
The growth factors such as Noggin, R-spondin and Wnt-3a, cost is relatively low, to be free of the intestinal stem cell of growth factor or crypts training
It supports；And culture medium according to embodiments of the present invention can be common to crypts of small intestine, colon crypt, unicellular and people's mouse small intestine
Crypts culture, is conveniently operated.What is more important, inventor have found, are trained using outside medium body according to embodiments of the present invention
Support intestinal crypts or intestinal stem cell, the quantity of the intestinal crypts of acquisition is more than the prior art, the ratio of the stem cell in the intestinal crypts of acquisition
Example higher, the intestinal stem cell of acquisition it is more；And utilize the culture medium in vitro culture intestines according to this year inventive embodiments
Crypts, can maintain the dryness of the stem cell in intestinal crypts for a long time, and cell Proliferation is more vigorous in intestinal crypts, thin in intestinal crypts
Born of the same parents' differentiation is normal orderly.Culture medium according to embodiments of the present invention is beneficial to gut epithelial stem cells culture systems and intestinal crypts organoid
The popularization of culture systems is conducive to intestinal stem cell and largely expands in vitro, and then provides and have very much for the treatment of intestines problem
The platform of profit.
According to an embodiment of the invention, above-mentioned culture medium can further include following additional technical feature at least it
One：
According to an embodiment of the invention, the concentration of the CHIR-99021 is 7~15 μM, the concentration of the LDN-193189
For 100~500nM.And then in vitro culture efficiency of the culture medium for intestinal crypts or intestinal stem cell further improves.
According to an embodiment of the invention, the culture medium further comprises blebbistatin.And then further promote intestines
The survival of stem cell.
According to an embodiment of the invention, the basal medium is Advanced DMEM/F12, optionally, the serum
Substitute is N2 and B27, optionally, described dual anti-for penicillin and streptomysin, optionally, the concentration of the glutamate/ester
For 2mM, optionally, the concentration of the N-acetylcystein is 1mM, and optionally, the concentration of the blebbistatin is 10 μ
M.And then preferably coordinative role can be played between the ingredient in culture medium, further improve the body of intestinal crypts or intestinal stem cell
Outer culture efficiency.
In the second aspect of the present invention, the present invention proposes CHIR-99021 and LDN-193189 in culture medium is prepared
Purposes, the culture medium is at least one of following：Intestinal crypts is cultivated, cultivates intestinal stem cell, promotes crypts of small intestine cell
Multiplication and/or differentiation.Inventor has found, CHIR-99021 and LDN-193189 can substitute EGF, Noggin, R-spondin and
The growth factors such as Wnt-3a, in the in-vitro culture medium of intestinal crypts or intestinal stem cell, so as to obtain the intestines without growth factor
Stem cell or crypts in-vitro culture medium；And CHIR-99021 and LDN-193189 substitute EGF, Noggin, R-spondin and
The culture medium that the growth factors such as Wnt-3a are obtained can be common to crypts of small intestine, colon crypt, unicellular and people's mouse small intestine
Crypts culture, significantly easy intestinal stem cell or crypts in vitro culture program.What is more important, inventor's discovery, CHIR-
99021 and LDN-193189 is substituted outside the medium body that the growth factors such as EGF, Noggin, R-spondin and Wnt-3a are obtained
Cultivate intestinal crypts or intestinal stem cell, the quantity of the intestinal crypts of acquisition more than the prior art, stem cell in the intestinal crypts of acquisition
Ratio higher, the intestinal stem cell of acquisition it is more；And utilization CHIR-99021 and LDN-193189 replacements EGF,
The culture medium in vitro culture intestinal crypts that the growth factors such as Noggin, R-spondin and Wnt-3a are obtained, can maintain for a long time
The dryness of stem cell in intestinal crypts, cell Proliferation is more vigorous in intestinal crypts, and cell differentiation is normally orderly in intestinal crypts.It utilizes
Culture medium prepared by CHIR-99021 and LDN-193189 is beneficial to gut epithelial stem cells culture systems and intestinal crypts organoid is trained
Support system popularization, be conducive to intestinal stem cell and largely expand in vitro, so for intestines problem treatment provide it is highly beneficial
Platform.
According to an embodiment of the invention, such use can further include at least one following additional technical feature：
According to an embodiment of the invention, the intestinal crypts is crypts of small intestine or colon crypt.Inventor has found, utilizes
CHIR-99021 and LDN-193189 substitutes the culture prepared by the growth factors such as EGF, Noggin, R-spondin and Wnt-3a
Base can be not only used for the in vitro culture of crypts of small intestine, can be also used for the in vitro culture of colon crypt, and the small intestine obtained is hidden
The quantity of nest or colon crypt organoid is significantly better than the prior art, and the quantity of wherein stem cell significantly increases, cell such as cup-shaped
Cell, the differentiation of secretory cell are normally orderly, and can maintain the dryness of stem cell in crypts for a long time.
According to an embodiment of the invention, the intestinal crypts is mouse intestinal crypts or people's intestinal crypts.Inventor has found, utilizes
CHIR-99021 and LDN-193189 substitutes the culture prepared by the growth factors such as EGF, Noggin, R-spondin and Wnt-3a
Base can be not only used for the in vitro culture of mouse small intestine crypts, can be also used for the in vitro culture of people's crypts of small intestine, incubation
The existence of middle people's crypts of small intestine and healthy growth are orderly, and very favorable put down is provided for the treatment of further human intestine's disease
Platform.
According to the embodiment that this year invents, the intestinal stem cell is small intestine epithelium stem cell or colon stem cell.Inventor
It was found that substitute the growth factors institutes such as EGF, Noggin, R-spondin and Wnt-3a using CHIR-99021 and LDN-193189
The culture medium of preparation can be not only used for the in vitro culture of crypts, can be also used for the unicellular in vitro culture of intestinal stem cell, institute
The single cells population of acquisition is substantially better than the prior art, the ratio of stem cell also showed increased.
In the third aspect of the present invention, the present invention proposes a kind of method for cultivating intestinal crypts.Implementation according to the present invention
Example, the described method includes：It will be cultivated in the intestinal crypts being wrapped in the matrigel in front culture medium.Utilize basis
The quantity for the intestinal crypts organoid that the method for this year inventive embodiments is obtained is significantly better than the number of the prior art, wherein stem cell
Amount significantly increases, and the differentiation of cell such as goblet cell, secretory cell is normally orderly, and can maintain to do in crypts for a long time thin
The dryness of born of the same parents.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature：
According to an embodiment of the invention, the intestinal crypts is crypts of small intestine or colon crypt.According to embodiments of the present invention
Method can be not only used for the in vitro culture of crypts of small intestine, can be also used for the in vitro culture of colon crypt.
According to an embodiment of the invention, the culture is in sterile, 5%CO2With 37 DEG C under conditions of carry out 1 week~2
Month.And then survival rate, the quantity of acquisition crypts organoid of crypts further improve.According to the method for the embodiment of the present invention not only
It can be used for the short time in vitro culture of crypts, such as 1 week, can be also used for the long-time in vitro culture of crypts, be such as up to 2
Month, the dryness of the stem cell in crypts remains unchanged for a long time.
In the fourth aspect of the present invention, the present invention proposes a kind of method for obtaining intestinal stem cell.Reality according to the present invention
Example is applied, the described method includes cultivated in the unicellular culture medium in front by intestinal stem cell or from intestinal crypts point
From acquisition, the intestinal crypts is obtained by foregoing method.The intestines obtained according to the method for the embodiment of the present invention
The quantity of stem cell is significantly better than the prior art.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature：
According to an embodiment of the invention, the intestinal stem cell is small intestine epithelium stem cell or colon stem cell.Utilize basis
The method of the embodiment of the present invention can be not only used for the unicellular culture of small intestine epithelium stem cell, can be also used for colon stem cell
Unicellular culture, the unicellular number showed increased obtained, the ratio of stem cell substantially increases.
According to an embodiment of the invention, the culture is in sterile, 5%CO2With 37 DEG C under conditions of carry out 1 week.And then
The single celled survival rate of stem cell, unicellular, stem cell the quantity of acquisition further improve.
It should be noted that the dryness of stem cell described herein refers to that stem cell dryness gene can be expressed, can break up
Various cell types are formed, individual cells have the ability for forming complete organoid, for example, intestinal stem cell described herein
Dryness refer to that intestinal stem cell dryness gene Lgr5 can be expressed, the various cell types of enteric epithelium can be differentiated to form, individual cells tool
Have to form the ability of complete intestines organoid.
In addition, it is necessary to explanation, the initial intestinal crypts of culture used in this application or intestinal stem cell be commercially available or
Voluntarily separation obtains, and separation method is those skilled in the art's routine experiment method, is not required to slap by surgical clinical training
It holds.
Description of the drawings
Fig. 1 is that 2ki systems according to embodiments of the present invention can maintain Lgr5- in mouse small intestine organoid in vitro
The dryness of GFP+ stem cells and the result figure of multiplication；
Fig. 2 is that 2ki systems according to embodiments of the present invention can be used for cultivating single Murine intestinal epithelium Lgr5-GFP+ and do
The result figure of cell；
Fig. 3 is that 2ki systems according to embodiments of the present invention can maintain Murine intestinal epithelium Lgr5-GFP+ to do carefully for a long time
The result figure of the dryness of born of the same parents；
Fig. 4 is secreting type cell in the Murine intestinal epithelium organoid that 2ki according to embodiments of the present invention is cultivated for a long time
Break up normal result figure；
Fig. 5 is that 2ki systems according to embodiments of the present invention can maintain stem cell in mouse Colon organoid in vitro
The result figure of dryness and cell proliferation and differentiation；And
Fig. 6 is the result figure for the crypts of small intestine in vitro culture that 2ki systems according to embodiments of the present invention can be used for people.
Specific embodiment
The embodiment of the present invention is described below in detail, the example of the embodiment is shown in the drawings.Below with reference to
The embodiment of attached drawing description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.
In the examples below, comprising basal medium Advanced DMEM/F12, serum substitute N2 and B27, double
The anti-, N-acetylcystein (N-acetylcysteine) of glutamate/ester (GlutaMAX) of 2mM, 1mM, 7-15 μM
The culture medium of the LDN-193189 of CHIR-99021 and 100-500nM；Or
Include Advanced DMEM/F12, serum substitute N2 and B27, dual anti-, 2mM glutamate/ester
(GlutaMAX), the N-acetylcystein (N-acetylcysteine) of 1mM, 7-15 μM CHIR-99021,100-500nM
The culture medium of LDN-193189 and 10 μM of blebbistatin be referred to as 2ki systems；
Existing crypts of small intestine culture system in vitro is referred to as ENR systems, comprising：Basal medium Advanced DMEM/
F12, serum substitute N2 and B27, the N-acetylcysteine of dual anti-, 2mM GlutaMAX and 1mM, the EGF of 50ng/mL,
The R-spondin 1 of the Noggin and 1ug/mL of 100ng/mL；
Existing intestinal stem cell culture system in vitro is referred to as ENRW systems, comprising：Advanced DMEM/F12, serum
Substitute N2 and B27, the N-acetylcysteine of dual anti-, 2mM GlutaMAX, 1mM, 10 μM of blebbistatin,
The R-spondin 1 of Noggin, 1ug/mL of EGF, 100ng/mL of 50ng/mL and the Wnt3a of 100ng/mL；
Existing colon crypt cultivating system is referred to as ENRW systems, comprising：Basal medium Advanced DMEM/
F12, serum substitute N2 and B27, dual anti-, 2mM GlutaMAX, 1mM N-acetylcysteine, 50ng/mL EGF,
The culture medium of the Wnt3a of the R-spondin 1 and 100ng/mL of Noggin, 1ug/mL of 100ng/mL；
Existing people's small intestine culture system in vitro is referred to as ENRW systems, comprising：Basal medium Advanced DMEM/
F12, serum substitute N2 and B27, the N-acetylcysteine of dual anti-, 2mM GlutaMAX, 1mM, 10 μM
The A83-01 of blebbistatin, 500nM, Gastrin I of 10 μM SB202190,10nM, 10mM Nicotinamide,
The culture medium of the Wnt3a of the R-spondin 1 and 100ng/mL of Noggin, 1ug/mL of EGF, 100ng/mL of 50ng/mL.
Mouse small intestine crypts and single stem cell culture of the embodiment 1 based on 2ki systems
Stem cell media according to 2ki provided by the invention is cultivating the purposes of stem cell, to being prepared in the present embodiment
Isolated mouse small intestine lacuna and mouse Lgr5+ small intestine adult stem cells cultivated.Using small intestine lacuna and Lgr5+ small intestines into
Somatic stem cell in vitro culture is small intestine organoid technology, has inquired into effects of the small molecule 2ki in this incubation.According to document
(Sato,T.et al.(2009).“Single Lgr5stem cells build crypt-villus structures in
Vitro without a mesenchymal niche. " Nature 459,262-5) report method, from Lgr5-EGFP-
IRES-creERT2 transgenic mices (can be organized to buy, Stock Number by The Jackson Laboratory:
008875, feature description：Green fluorescence protein gene GFP is manually inserted into the promoter downstream of endogenous Lgr5 genes, therefore
The cell that Lgr5 genes are expressed in this transgenic mice is marked by green fluorescent protein GFP, positive available for tracking Lgr5
Adult stem cell) in separate and cultivate small intestine lacuna and Lgr5+ small intestine adult stem cells, 2ki is in vitro culture survival rate for observation
With the influence of the speed of growth, specific method is as described below：
(1) small intestine is isolated from Lgr5-EGFP-IRES-creERT2 transgenic mices, by its rip cutting and with precooling
The PBS buffer solution that pH value is 7.4 is washed three times, to remove chyme, carefully scrapes off the small intestine suede on small intestine inner wall with scalpel afterwards
Hair.By remaining Partial Shear into the intestines piece of 5mm length and with the PBS buffer solution containing 10mM EDTA in 40 points of incubation on ice
Clock.The PBS buffer solution containing EDTA is changed with the PBS buffer solution of fresh precooling, wherein with pipettor and the pipette of 10ml
Intestines piece is tempestuously blown and beaten repeatedly, and massive small bowel lacuna is made to be fallen to from intestines on piece in supernatant.Obtained supernatant is thin with 70 μm of apertures
Born of the same parents sieve (being purchased from BD companies, article No. 352350) filtering, and with the intestinal villi of removal remaining, 3 points are centrifuged using 600rpm rotating speeds
Clock removal is scattered unicellular.More pure small intestine lacuna is precipitated as after centrifugation, available in vitro culture and by rear
Continuous processing therefrom obtains Lgr5+ small intestine adult stem cells.
(2) by every 100 separated small intestine lacunas and the matrigel of 50 microlitres of precoolings (being purchased from BD companies, article No. 354230)
It is laid in after mixing in a hole of 48 porocyte culture plates.Culture plate is placed in room temperature 20 minutes, package small intestine is allowed to fall into
The matrigel of nest fully solidifies.Control group use containing basal medium Advanced DMEM/F12, serum substitute N2 and
B27, the N-acetylcysteine of dual anti-, 2mM GlutaMAX and 1mM and required growth factor：The EGF of 50ng/mL,
Culture medium (ENR) culture of the R-spondin 1 of the Noggin and 1ug/mL of 100ng/mL.2ki experimental groups are used containing basis
Culture medium A dvanced DMEM/F12, serum substitute N2 and B27, the N- of dual anti-, 2mM GlutaMAX and 1mM
The LDN- of acetylcysteine and 10uM CHIR-99021 (purchased from Selleck companies or MCE companies) and 200nM
193189 cultures (are purchased from Selleck companies).After being cultivated 7 days in carbon dioxide cell incubator, survived with micro- sem observation
Small intestine lacuna and count, experimental result is as shown in Figure 1A.Experimental group by 2ki cultures and the control group by ENR cultures,
No significant difference in the form of organoid cultures.Statistics further shows that the organoids quantity of 2ki cultures is substantially more
In control group.Secondly we observe the stem cell of the GFP positives using fluorescence microscope and use flow cytometer
(MoFlo XDP, Beckman) analyzes GFP positive cells, as a result as shown in Figure 1B, in the organoid of 2ki cultures
The ratio of Lgr5-GFP+ stem cells is higher than the organoid that ENR is cultivated.
(3) then dyeing, specific operation process is marked to the cell of multiplication using EdU (Invitrogen) again in we
It is as follows.Illustrate to operate according to EdU (Invitrogen), first to the organoids under normal culture conditions and 2ki condition of culture
Carry out EdU marks, add in the EdU of 10uM, be put into 37 DEG C of culture mediums continue culture 2 it is small when.After discarding culture medium, delayed using PBS
Rinse is resuspended twice in fliud flushing, is placed at room temperature for one hour using 4% paraformaldehyde and is fixed.Organoids after fixation is used
Rinse is resuspended twice in 5%BSA, and penetrating three hours of rocking-turn in cabinet is chromatographed at 4 DEG C using 2%Triton.Reuse 5%BSA weights
After outstanding rinse, reaction substrate is prepared to specifications and is added in, incubation at room temperature carries out being protected from light dyeing for one hour.Rinse is resuspended in 5%BSA
DAPI dyeing and mounting are carried out afterwards.Afterwards observation of taking pictures is carried out using laser confocal microscope (Olympus).Such as the knot of Fig. 1 C
Shown in fruit, the cell Proliferation in the organoid of ENR and 2ki cultures does not have significant difference.It is above-mentioned the experimental results showed that, 2ki can be with
Effectively growth factor is replaced to carry out in vitro culture to crypts of small intestine, while the survival rate and dryness of small intestine organoid can be improved.
(4) the small intestine lacuna Tryple obtained will be separated from Lgr5-EGFP-IRES-creERT2 transgenic mices
(being purchased from Invitrogen companies) handles half an hour in 37 degree, then is blown and beaten 10 times or more with the syringe of 1.2mm, and small intestine is fallen into
Nest digestion is broken up to be unicellular.Obtained supernatant is filtered with the cell sieve (being purchased from BD companies, article No. 352340) in 40 μm of apertures, to go
Except the cell mass of remaining, and sort (purchased from BD companies, model FACSAria II) with flow cytometer that obtain its Green glimmering
The Lgr5+ small intestine adult stem cells of signal.Obtained Lgr5+ small intestines adult stem cell is sorted by every 5000 and 50 microlitres pre-
Cold matrigel is laid in after mixing in a hole of 48 porocyte culture plates.After matrigel fully solidifies, control group makes
With containing basal medium Advanced DMEM/F12, serum substitute N2 and B27, dual anti-, 2mM GlutaMAX and 1mM
N-acetylcysteine, 10 μM of blebbistatin and required growth factor：EGF, 100ng/mL's of 50ng/mL
Culture medium (ENRW) culture of the Wnt3a of the R-spondin 1 and 100ng/mL of Noggin and 1ug/mL.2ki experimental groups use
Containing basal medium Advanced DMEM/F12, serum substitute N2 and B27, dual anti-, 2mM GlutaMAX and 1mM N-
The LDN-193189 cultures of acetylcysteine, 10 μM of blebbistatin and 10 μM of CHIR-99021 and 100nM.
After being cultivated 7 days in carbon dioxide cell incubator, the organoid with the unicellular formation of micro- sem observation and the class device to formation
Official is counted, and as shown in Figure 2 A, the unicellular number formed in 2ki systems is significantly more than the Dan Ke in ENRW to experimental result
It is grand.Afterwards using fluorescence microscope take pictures with flow cytometry analysis obtain as shown in Figure 2 B as a result, 2ki culture clone in
The ratio of stem cell is also significantly more than the clone of ENRW cultures.
Mouse small intestine crypts long-term cultivation of the embodiment 2 based on 2ki systems
It is small intestine organoid technology using small intestine lacuna and Lgr5+ small intestine adult stem cells in vitro culture, has inquired into small point
Effects of the sub- 2ki in this incubation.Then, we determine the cultivating system not with the system have been long-term cultivation
It can influence crypts of small intestine and the various functions and characteristic of stem cell.Specific method is as described below：
(1) small intestine is isolated from Lgr5-EGFP-IRES-creERT2 transgenic mices, by its rip cutting and with precooling
The PBS buffer solution that pH value is 7.4 is washed three times, to remove chyme, carefully scrapes off the small intestine suede on small intestine inner wall with scalpel afterwards
Hair.By remaining Partial Shear into the intestines piece of 5mm length and with the PBS buffer solution containing 10mM EDTA in 40 points of incubation on ice
Clock.The PBS buffer solution containing EDTA is changed with the PBS buffer solution of fresh precooling, wherein with pipettor and the pipette of 10ml
Intestines piece is tempestuously blown and beaten repeatedly, and massive small bowel lacuna is made to be fallen to from intestines on piece in supernatant.Obtained supernatant is thin with 70 μm of apertures
Born of the same parents sieve (being purchased from BD companies, article No. 352350) filtering, and with the intestinal villi of removal remaining, 3 points are centrifuged using 600rpm rotating speeds
Clock removal is scattered unicellular.More pure small intestine lacuna is precipitated as after centrifugation, available in vitro culture and by rear
Continuous processing therefrom obtains Lgr5+ small intestine adult stem cells.
(2) by every 100 separated small intestine lacunas and the matrigel of 50 microlitres of precoolings (being purchased from BD companies, article No. 354230)
It is laid in after mixing in a hole of 48 porocyte culture plates.Culture plate is placed in room temperature 20 minutes, package small intestine is allowed to fall into
The matrigel of nest fully solidifies.Control group use containing basal medium Advanced DMEM/F12, serum substitute N2 and
B27, the N-acetylcysteine of dual anti-, 2mM GlutaMAX and 1mM and required growth factor：The EGF of 50ng/mL,
Culture medium (ENR) culture of the R-spondin 1 of the Noggin and 1ug/mL of 100ng/mL.2ki experimental groups are used containing basis
Culture medium A dvanced DMEM/F12, serum substitute N2 and B27, the N- of dual anti-, 2mM GlutaMAX and 1mM
The LDN- of acetylcysteine and 10uM CHIR-99021 (purchased from Selleck companies or MCE companies) and 200nM
193189 cultures (are purchased from Selleck companies).It is cultivated 60 days in carbon dioxide cell incubator, during which according to the shape of organoid
State is passed on, and the small intestine lacuna finally survived with micro- sem observation simultaneously counts, and experimental result is as shown in Figure 3A.It is cultivated by 2ki
Experimental group with by ENR cultivate control group, the no significant difference in the form of organoid.Statistics is further shown
The organoids quantity of 2ki cultures is significantly more than control group.Secondly we use stem cell of the fluorescence microscope to the GFP positives
It is observed and flow cytometer (MoFlo XDP, Beckman) is used to analyze GFP positive cells, as a result such as Fig. 3 B institutes
Show, the organoid of the brightness ratio ENR cultures of Lgr5-GFP+ stem cells is high in the organoid of 2ki cultures, and statistics is also the same
The ratio of the organoid Lgr5-GFP stem cells of 2ki cultures is shown also apparently higher than control group.
(2) it is normal to further prove the organoid of longterm culture in vitro, is not mutated.We are to culture 60
It organoid is with Tryple (being purchased from Invitrogen companies) in 37 degree of digestion process half an hours, then the injection with 1.2mm
Device is blown and beaten 10 times or more, and the digestion of small intestine lacuna is broken up to be unicellular.Obtained supernatant (is purchased from BD public affairs with the cell sieve in 40 μm of apertures
Department, article No. 352340) filtering, with the cell mass of removal remaining, and with flow cytometer (purchased from BD companies, model
FACSAria II) sorting obtain the Lgr5+ small intestine adult stem cells of wherein green fluorescent label.It collects by ENR cultures and 2ki
The stem cell each 200,000 of culture carries out RNA extractions using RNA extracts kits (being purchased from TIAGEN, article No. 74104).Through measurement
After RNA is errorless, meets at Nuo He companies and carry out transcript profile large scale sequencing.After obtaining sequencing result, the biology between sample is carried out
Informatics compares, and as shown in Figure 3 C, by external long-term culture, the organoid of ENR and 2ki cultures is normal, is not sent out
Raw any mutation.At the same time, we have also carried out the karyotyping of organoid, and it is peace to further demonstrate 2ki culture systems
It is complete effective, with long-term cultivation and the normal condition of small intestine organoid can be maintained.
(3) we by a series of cell experiment prove long-term cultivation stem cell be have normal self-renewing and
Differentiation capability.The organoid that we cultivate ENR and 2ki 60 days is disappeared with Tryple (being purchased from Invitrogen companies) in 37 degree
Change processing half an hour, then blown and beaten 10 times or more with the syringe of 1.2mm, the digestion of small intestine lacuna is broken up to be unicellular.It is obtained
Supernatant is filtered with the cell sieve (being purchased from BD companies, article No. 352340) in 40 μm of apertures, with the cell mass of removal remaining, and uses streaming
Cell instrument (purchased from BD companies, model FACSAria II) sorting obtains the Lgr5+ small intestines of wherein green fluorescent label into soma
Cell.Obtained Lgr5+ small intestines adult stem cell is sorted by every 5000 and the matrigel of 50 microlitres of precoolings is put down after mixing
It is laid in a hole of 48 porocyte culture plates.After matrigel fully solidifies, two groups using containing basal medium
Advanced DMEM/F12, serum substitute N2 and B27, the N- of dual anti-, 2mM GlutaMAX and 1mM
Acetylcysteine, 10 μM of blebbistatin and required growth factor：EGF, 100ng/mL's of 50ng/mL
Two kinds long-term training is observed in culture medium (ENRW) culture of the Wnt3a of the R-spondin 1 and 100ng/mL of Noggin and 1ug/mL
Whether the dryness and self-renewal capacity of foster stem cell are variant.After being cultivated 7 days in carbon dioxide cell incubator, with aobvious
Micro mirror is observed survival rate and is counted, and experimental result can form comparable unicellular number as shown in Figure 3D, in ENR and 2ki systems.
Statistical analysis afterwards as shown in Figure 3D as a result, clone's number indifference of clone's number and ENR culture of 2ki cultures.
(4) for the organoid of long-term cultivation, the differentiation of these organoids is proved in the dyeing We conducted noble cells
Cell is normal.We fix one hour with 4% paraformaldehyde to the ENR and 2ki organoids cultivated 60 days in room temperature.PBS
After rinse three times is resuspended in buffer solution, penetrating three hours of rocking-turn in 4 DEG C of chromatography cabinets are placed on using 2%Triton.Then use 3%
After BSA and 1%Triton room temperatures are closed three hours, Lyz is added in, after Muc2 and Chga antibody (being purchased from Santa-Cruz companies)
4 degree of overnight incubations, PBS rinses are three times.Room temperature stain incubation is carried out using the rabbit secondary antibody and DAPI of 488- couplings one hour, then
It is observed using PBS rinses and mounting.As shown in figure 4, the paneth's cell of the EdU proliferative conditions of the two, Lyz characterization, Muc2 characterizations
Goblet cell and Chga characterization small enteroendocrine cell be indifference, illustrate two cultivating systems turn out class device
The basic indifference of official's noble cells.At the same time, we are further verified using real-time fluorescence quantitative PCR, wherein Alpi characterizations
Enterocyte lowered may be due to 2ki there may be the facilitation to stem cell and noble cells inhibition work
With.
Mouse Colon crypts in vitro culture of the embodiment 3 based on 2ki systems
Stem cell media according to 2ki provided by the invention is cultivating the purposes of stem cell, to being prepared in the present embodiment
Isolated mouse colon lacuna cultivated.It is small intestine organoid technology using colon lacuna in vitro culture, has inquired into small molecule
Effects of the 2ki in this incubation.According to document (Sato, T.et al. (2011) " Long-term Expansion of
Epithelial Organoids From Human Colon,Adenoma,Adenocarcinoma,and Barrett’s
Epithelium.”Gastroenterology 141:1762-1772) the method for report, from Lgr5-EGFP-IRES-
CreERT2 transgenic mices (can be organized to buy, Stock Number by The Jackson Laboratory:008875,
Feature describes：Green fluorescence protein gene GFP is manually inserted into the promoter downstream of endogenous Lgr5 genes, so transgenosis is small
The cell that Lgr5 genes are expressed in mouse is marked by green fluorescent protein GFP, the adult stem cell available for the tracking Lgr5 positives)
It is middle to separate and cultivate colon lacuna, influences of the observation 2ki in vitro culture survival rate and the speed of growth, the following institute of specific method
It states：
(1) colon is isolated from Lgr5-EGFP-IRES-creERT2 transgenic mices, by its rip cutting and with precooling
The PBS buffer solution that pH value is 7.4 is washed three times, to remove chyme.By remaining Partial Shear into the intestines piece of 5mm length and with containing
The PBS buffer solution of 10mM EDTA is incubated 40 minutes on ice.The PBS containing EDTA is changed with the PBS buffer solution of fresh precooling to delay
Fliud flushing tempestuously blows and beats intestines piece with the pipette of pipettor and 10ml wherein, a large amount of colon lacunas is made to be taken off from intestines on piece repeatedly
It falls in supernatant.Obtained supernatant is filtered with the cell sieve (being purchased from BD companies, article No. 352350) in 70 μm of apertures, using 600rpm
Rotating speed centrifugation removal in 3 minutes is scattered unicellular.More pure colon lacuna is precipitated as after centrifugation, available in vitro culture
And Lgr5+ colon adult stem cells are therefrom obtained by subsequent processing.
(2) by every 100 separated colon lacunas and the matrigel of 50 microlitres of precoolings (being purchased from BD companies, article No. 354230)
It is laid in after mixing in a hole of 48 porocyte culture plates.Culture plate is placed in room temperature 20 minutes, package colon is allowed to fall into
The matrigel of nest fully solidifies.Control group use containing basal medium Advanced DMEM/F12, serum substitute N2 and
B27, the N-acetylcysteine of dual anti-, 2mM GlutaMAX and 1mM and required growth factor：The EGF of 50ng/mL,
Culture medium (ENRW) culture of the Wnt3a of the R-spondin 1 and 100ng/mL of Noggin, 1ug/mL of 100ng/mL.2ki is real
Group use is tested containing basal medium Advanced DMEM/F12, serum substitute N2 and B27, dual anti-, 2mM GlutaMAX
With N-acetylcysteine the and 10uM CHIR-99021 of 1mM (purchased from Selleck companies or MCE companies) and
The LDN-193189 cultures (being purchased from Selleck companies) of 200nM.After being cultivated 7 days in carbon dioxide cell incubator, use is micro-
The colon lacuna of sem observation survival simultaneously counts, and experimental result is as shown in Figure 5A.By the experimental group of 2ki cultures and by ENRW cultures
Control group, the no significant difference in the form of organoid cultures.Statistics further shows the organoids of 2ki cultures
Quantity is significantly more than control group.Secondly we observe the stem cell of the GFP positives using fluorescence microscope and use streaming
Cell instrument (MoFlo XDP, Beckman) analyzes GFP positive cells, as a result as shown in Figure 5 C, the organoid of 2ki cultures
The ratio of middle Lgr5-GFP+ stem cells is higher than the organoid that ENRW is cultivated.
(3) then dyeing, specific operation process is marked to the cell of multiplication using EdU (Invitrogen) again in we
It is as follows.Illustrate to operate according to EdU (Invitrogen), first to the organoids under normal culture conditions and 2ki condition of culture
Carry out EdU marks, add in the EdU of 10uM, be put into 37 DEG C of culture mediums continue culture 2 it is small when.After discarding culture medium, delayed using PBS
Rinse is resuspended twice in fliud flushing, is placed at room temperature for one hour using 4% paraformaldehyde and is fixed.Organoids after fixation is used
Rinse is resuspended twice in 5%BSA, and penetrating three hours of rocking-turn in cabinet is chromatographed at 4 DEG C using 2%Triton.Reuse 5%BSA weights
After outstanding rinse, reaction substrate is prepared to specifications and is added in, incubation at room temperature carries out being protected from light dyeing for one hour.Rinse is resuspended in 5%BSA
DAPI dyeing and mounting are carried out afterwards.Afterwards observation of taking pictures is carried out using laser confocal microscope (Olympus).Such as the knot of Fig. 5 B
Shown in fruit, the cell Proliferation in the organoid of ENRW and 2ki cultures does not have significant difference.It is above-mentioned the experimental results showed that, 2ki can be with
Effectively growth factor is replaced to carry out in vitro culture to colon crypt, while the survival rate and dryness of colon organoid can be improved.
(4) for the colon organoid of culture, the differentiation of these organoids is proved in the dyeing We conducted noble cells
Cell is normal.We fix one hour with 4% paraformaldehyde to the ENRW and 2ki organoids cultivated in room temperature.PBS is buffered
After rinse three times is resuspended in liquid, penetrating three hours of rocking-turn in 4 DEG C of chromatography cabinets are placed on using 2%Triton.Then use 3%BSA
After being closed three hours with 1%Triton room temperatures, Muc2 antibody (being purchased from Santa-Cruz companies) 4 degree of overnight incubations afterwards are added in,
PBS rinses are three times.Room temperature stain incubation is carried out using the rabbit secondary antibody and DAPI of 488- couplings one hour, then uses PBS rinses
And mounting is observed.As shown in figure 4, the goblet cell of the Muc2 characterizations of the two is indifference, illustrate two cultivating system cultures
The basic indifference of organoid noble cells out.
The enteric epithelium vitro culture system for the people that embodiment 4 is established based on 2ki systems
Stem cell media according to 2ki provided by the invention is cultivating the purposes of stem cell, to being prepared in the present embodiment
In vitro people's small intestine lacuna cultivated.It is as follows：
The 1st, the size intestinal segment that surgical operation intercepts is immediately placed in the pH value of precooling in 7.4 PBS buffer solution, to rinse three
All over to remove bloodstain.The normal intestinal epithelial tissue of lesion not yet occurs in broken-hearted middle searching, and passes through mechanical means by itself and bag
Fat is included to separate with its hetero-organization including muscle layer.The intestinal villus on enteral wall is carefully scraped off with scalpel afterwards.By residue
Partial Shear into 5mm length intestines piece and with the PBS buffer solution containing 10mM EDTA on ice be incubated 40 minutes.With fresh
The PBS buffer solution of precooling changes the PBS buffer solution containing EDTA, wherein with the pipette of pipettor and 10ml tempestuously repeatedly
Intestines piece is blown and beaten, a large amount of intestines lacunas is made to be fallen to from intestines on piece in supernatant.Obtained supernatant is filtered with the cell sieve in 70 μm of apertures, to go
Except the intestinal villus of remaining, it is scattered using 600rpm rotating speeds centrifugation removal in 3 minutes unicellular.Being precipitated as after centrifugation is more pure
Net intestines lacuna, available in vitro culture.The obtaining means of sample and follow-up scientific research scheme have been entrusted by chain hospital ethics
Member can audit.
2nd, the matrigel of every 100 separated small intestines and 50 microlitres of precoolings is laid in 48 hole cell culture after mixing
In one hole of plate.After matrigel fully solidifies, control group is used containing basal medium Advanced DMEM/F12, serum
The N-acetylcysteine of substitute N2 and B27, dual anti-, 2mM GlutaMAX and 1mM, 10 μM of blebbistatin,
The Nicotinamide of the Gastrin I and 10mM of the A83-01 of 500nM, 10 μM of SB202190,10nM and required growth
The factor：The training of the Wnt3a of the R-spondin 1 and 100ng/mL of the Noggin and 1ug/mL of EGF, 100ng/mL of 50ng/mL
Support base (ENRW) culture.2ki experimental groups use containing basal medium Advanced DMEM/F12, serum substitute N2 and
The N-acetylcysteine of B27, dual anti-, 2mM GlutaMAX and 1mM, 10 μM of blebbistatin and 10uM
The LDN-193189 cultures of CHIR-99021 and 100nM.And with micro- sem observation survive small intestine lacuna and count.The results are shown in
In Fig. 6.
As shown in fig. 6, in experimental group by 2ki culture in vitro people's crypts of small intestine in vitro culture survival quantity with compareing
The crypts indifference that ENRW is cultivated in group.The experimental results showed that 2ki can effectively replace growth factor in vitro people's small intestine lacuna
Carry out in vitro culture.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms is not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It is combined in an appropriate manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the different embodiments described in this specification or example and different embodiments or exemplary feature
It closes and combines.
Although the embodiment of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (10)
1. a kind of culture medium, which is characterized in that including：Basal medium, serum substitute, dual anti-, glutamate/ester, N- second
Acyl cysteine, CHIR-99021 and LDN-193189.
2. culture medium according to claim 1, which is characterized in that the concentration of the CHIR-99021 is 7~15 μM, described
The concentration of LDN-193189 is 100~500nM,
Optionally, the culture medium further comprises blebbistatin.
3. culture medium according to claim 2, which is characterized in that the basal medium is Advanced DMEM/F12,
Optionally, the serum substitute is N2 and B27,
It is optionally, described dual anti-for penicillin and streptomysin,
Optionally, the concentration of the glutamate/ester is 2mM,
Optionally, the concentration of the N-acetylcystein is 1mM,
Optionally, the concentration of the blebbistatin is 10 μM.
Purposes of the 4.CHIR-99021 and LDN-193189 in culture medium is prepared, the culture medium for it is following at least it
One：
Cultivate intestinal crypts,
Cultivate intestinal stem cell,
Promote crypts of small intestine cell Proliferation and/or differentiation,
Optionally, the intestinal crypts be crypts of small intestine or colon crypt,
Optionally, the intestinal crypts be mouse intestinal crypts or people's intestinal crypts,
Optionally, the intestinal stem cell is small intestine epithelium stem cell or colon stem cell.
A kind of 5. method for cultivating intestinal crypts, which is characterized in that including：By the intestinal crypts being wrapped in matrigel in claim
It is cultivated in 1~3 any one of them culture medium.
6. according to the method described in claim 5, it is characterized in that, the intestinal crypts is crypts of small intestine or colon crypt.
7. according to the method described in claim 5, it is characterized in that, the culture is in sterile, 5%CO2Under conditions of 37 DEG C
It carries out 1 week~2 months.
A kind of 8. method for obtaining intestinal stem cell, which is characterized in that by the unicellular any in claims 1 to 3 of intestinal stem cell
It is cultivated or is separated from intestinal crypts in culture medium described in and obtained, the intestinal crypts is any by claim 5~7
What the method described in obtained.
9. according to the method described in claim 8, it is characterized in that, the intestinal stem cell is done for small intestine epithelium stem cell or colon
Cell.
10. according to the method described in claim 8, it is characterized in that, the culture is in sterile, 5%CO2With 37 DEG C of condition
It is lower to carry out 1 week.
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