CN106367382A - Culture medium for promoting in-vitro expansion of pig intestinal tract pit cells and culture method thereof - Google Patents
Culture medium for promoting in-vitro expansion of pig intestinal tract pit cells and culture method thereof Download PDFInfo
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Abstract
The invention discloses a culture medium for promoting in-vitro expansion of pig intestinal tract pit cells. The culture medium is prepared from an Advanced DMEM/F12 culture solution which contains 100-200 ng/mL Wnt3a, 1%-2% N2, 1%-2% B27, 0.5-1 mmol/L N-acetyl-cysteine, 1-2 mmol/L L-glutamine, 5-10 micromol/L SB202190, 0.5-1 micromol/L LY2157299, 50-100 ng/mL EGF, 100-200 ng/mL Noggin and 500-1,000 ng/mL R-Spondin 1. On the basis of component screening and content optimization, the culture medium can be successfully used for in-vitro culture of pig intestinal tract pit single cells; in addition, by preparing the CDX2 conditioned culture medium, the dosage and using method of the culture medium in pig intestinal tract pit cell culture are optimized, so that the purpose of promoting in-vitro expansion of the pig intestinal tract pit cells is achieved.
Description
Technical field
The present invention relates to technical field of cell culture, Intestinum Sus domestica road pit cell is promoted to expand in vitro more particularly, to a kind of
The culture medium increasing and its cultural method.
Background technology
Intestinal is not only the main place of mammalian nutrition substance digestion absorption, is also the maximum immunity of body and interior point
Secrete organ, have concurrently digest and assimilate, function that immune defence and nerve-endocrine are adjusted, its material base is in quickly updating
The enteric epithelium of state.The crypt-villus axle that gut epithelium is connected by structure is constituted, and is divided into functional areas and breeding blanket, breeds position
In crypts position, it is the power source that enteric epithelium updates.At present, still lack Intestinum Sus domestica road pit cell extracorporeal culturing method.
Content of the invention
The technical problem to be solved is the drawbacks described above overcoming prior art to exist, and provides a kind of promotion Intestinum Sus domestica
The culture medium of road pit cell amplification in vitro.
Second object of the present invention is to provide a kind of cultural method promoting Intestinum Sus domestica road pit cell amplification in vitro.
The purpose of the present invention is achieved by the following technical programs:
A kind of culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro, the consisting of of described culture medium: advanced dmem/
In f12 culture fluid contain 100～200 ng/ml wnt3a, 1～2% n2,1～2% b27,0.5～1 mmol/l n- acetyl-
Cysteine, 1～2 mmol/l l- L-Glutamine, 5～10 μm of ol/l sb202190,0.5～1 μm of ol/l
Ly2157299,50～100 ng/ml egf, 100～200 ng/ml noggin and 500～1000 ng/ml r-spondin
1.
The present invention first passes through the screening of component and the optimization of content, obtains one kind and can promote Intestinum Sus domestica road pit cell
The basal medium of amplification in vitro.Wnt3a in this culture medium can be combined with the frizzled family receptors of cell surface, raises
Collection lrp5/6, makes the stable beta-catenin protein level of intracellular rise, stable beta-catenin and tcf/lef transcribes
The factor combines, thus regulating and controlling the expression of wnt target gene, is conducive to the normal development of enterocyte；R-spondin 1 can be competitive
Ground is combined with receptor kremen and lrp-6 of wnt, and suppression dkk-1 mediates the internalization of wnt receptor, thus activating wnt/beta-
Catenin signal；L- L-Glutamine is the main energy substance of intestinal epithelial cell and important function regulator；Noggin is
The antagonist of bmp-4 albumen, can be combined with tgf- β, thus suppressing its signal transduction；N- acetyl-cysteine plays antioxidation
Effect；These interaction between component above-mentioned, reach the effect of amplification Intestinum Sus domestica road pit cell jointly.
In addition, by preparing cdx2 Conditioned immunolresponse, and find for the research of Intestinum Sus domestica road pit cell amplification in vitro,
Cdx2 culture medium can remarkably promote Intestinum Sus domestica road pit cell amplification in vitro.Therefore, the present invention also provides another to promote pig
The culture medium (also referred to as Optimal Medium) of intestinal pit cell amplification in vitro, the composition of this Optimal Medium is by basis culture
Advanced dmem/f12 culture fluid in base replaces with cdx2 conditioned medium, remaining composition and the content of 15～50 v/v%
Identical with basal medium.
Therefore, the consisting of of the described culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro: cdx2 conditioned medium,
100～200ng/ml wnt3a, 1～2% n2,1～2% b27,0.5～1 mmol/l n- acetyl-cysteine, 1～2
Mmol/l l- L-Glutamine, 5～10 μm of ol/l sb202190,0.5～1 μm of ol/l ly2157299,50～100 ng/ml
Egf, 100～200 ng/ml noggin and 500～1000 ng/ml r-spondin 1；Described cdx2 conditioned medium is in institute
Stating the volume fraction in the culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro is 15～50 v/v%；Described cdx2 condition training
Foster base is obtained by following steps: (1) will be a number of, and the cell containing cdx2 gene (described is trained completely in complete medium
Foster base is to contain 5～10% fbs, 0.5～1% penicillin/streptomycin, 1～2 mmol/ in advanced dmem/f12 culture fluid
L l- L-Glutamine and 5～10 mmol/l hepes) in culture 3.5～4 days after, collect supernatant, after filtration filtrate 1；
(2) continue the cell containing cdx2 gene for the culture, after culture 2～3 days, collect supernatant again, filter to get filtrate 2, by filtrate 1
Obtain cdx2 Conditioned immunolresponse with filtrate 2 by 1:1～0.8:1 volume mixture.
Cdx2(caudal type homeobox transcription factor 2) it is gut epithelium specific transcriptional
The factor, scalable Sucrase-isomaltase (sucrase-isomaltase, si), Lactose enzyme-Phlorizin hydrolase
(lactase phlorizin hydrolase, lph), goblet cell specific gene mucin 2 (muc 2), small peptide transhipment carry
Body pept1, cationic amino acid transporter y+The expression of the functional genes such as lat1, to intestinal growth, intestinal epithelial cell break up,
Propagation has important adjustment effect.
Wherein, described cdx2 conditioned medium is obtained by following steps: (1) will be a number of, containing cdx2 gene
Cell complete medium (complete medium be in advanced dmem/f12 culture fluid contain 5～10% fbs, 0.5～1%
Penicillin/streptomycin, 1～2 mmol/l l- L-Glutamine and 5～10 mmol/l hepes) in culture 3.5～4 days after, receive
Collection supernatant, obtains filtrate 1 after filtration；(2) continue the cell containing cdx2 gene for the culture, after cultivating 2～3 days, collect again
Clear liquid, filters to get filtrate 2, and filtrate 1 and filtrate 2 are obtained cdx2 Conditioned immunolresponse by 1:1～0.8:1 volume mixture.
Preferably, can above-mentioned any one promote Intestinum Sus domestica road pit cell amplification in vitro culture medium in add 1～
2% penicillin/streptomycin or/and 5～10 mmol/l hepes or/and 5～10 mmol/l nicotine, are more beneficial for cell culture.
The present invention also provides a kind of method promoting Intestinum Sus domestica road pit cell amplification in vitro, is unicellular in Intestinum Sus domestica road crypts
The middle culture medium adding any one promotion Intestinum Sus domestica road pit cell amplification in vitro above-mentioned is cultivated.
The present invention also provides the cultural method of another Intestinum Sus domestica road pit cell, is in culture early stage, hidden in Intestinum Sus domestica road
The unicellular middle addition basal medium of nest is cultivated；Late stage of culture is replaced by Optimal Medium and is cultivated.
Compared with prior art, the method have the advantages that
The invention provides a kind of culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro, the consisting of of described culture medium:
In advanced dmem/f12 culture fluid contain 100～200 ng/ml wnt3a, 1～2% n2,1～2% b27,0.5～1
Mmol/l n- acetyl-cysteine, 1～2 mmol/l l- L-Glutamine, 5～10 μm of ol/l sb202190,0.5～1 μ
Mol/l ly2157299,50～100 ng/ml egf, 100～200 ng/ml noggin and 500～1000 ng/ml r-
spondin 1；By the screening of component and the optimization of content, this culture medium can be used successfully to the single celled body of Intestinum Sus domestica road crypts
Outer culture, simultaneously on the basis of basal medium, by preparing cdx2 Conditioned immunolresponse, optimizes it thin in Intestinum Sus domestica road crypts
Consumption in born of the same parents' culture and using method, to reach the purpose promoting Intestinum Sus domestica road pit cell amplification in vitro.
Brief description
Fig. 1 is the morphologic observation of the basic-ipec-j2 and cdx2-ipec-j2 cell strain that embodiment 1 obtains, wherein, figure
1a is basic-ipec-j2 cell, and Fig. 1 b is cdx2-ipec-j2 cell.
Fig. 2 detects, for real time fluorescent quantitative pcr, basic-ipec-j2 the and cdx2-ipec-j2 cell that embodiment 1 obtains
The expression of cdx2 in strain.
Fig. 3 detects, for western blotting, basic-ipec-j2 the and cdx2-ipec-j2 cell that embodiment 1 obtains
The expression of cdx2 in strain.
Fig. 4 is the pit cell microscope figure of embodiment 6 methods described culture.
Fig. 5 is the aobvious of the pit cell of Optimal Medium 1 culture using the basal medium of embodiment 6 and embodiment 8
Micro mirror figure, wherein, Fig. 5 a is the pit cell of the base culture base using embodiment 6；Fig. 5 b is using Optimal Medium 1
The pit cell of culture.
Fig. 6 utilizes the micro- of the pit cell of Optimal Medium 2 culture of the basal medium of embodiment 6 and embodiment 9
Mirror figure, wherein, Fig. 6 a is the pit cell of the base culture base using embodiment 6；Fig. 6 b is to be trained using Optimal Medium 2
Foster pit cell.
Fig. 7 is the microscope figure of the pit cell that experimental group 1 described in embodiment 10 arrives experimental group 3 culture, and Fig. 7 a is experiment
The pit cell of group 1 culture；Fig. 7 b is the pit cell of experimental group 2 culture；Fig. 7 c is the pit cell of experimental group 3 culture.
Fig. 8 is the microscope figure of the pit cell of comparative example 1 culture.
Fig. 9 is the microscope figure of the pit cell of comparative example 2 culture.
Specific embodiment
Further illustrate present disclosure below in conjunction with Figure of description and specific embodiment, but should not be construed as right
The restriction of the present invention；In the case of present invention spirit and essence, modification that the inventive method, step, condition are made
Or replace, belong to the scope of the present invention.Unless otherwise noted, experimental technique used in embodiment is people in the art
Conventional method known to member and technology, reagent or material are and are obtained by commercial sources.
Embodiment 1 obtains the ipec-j2 cell strain of overexpression cdx2
Complete medium used by the present embodiment is: dmem+10% fbs.
With lipofectamin3000(invitrogen) respectively by basic-pcdna3.1+ and cdx2-pcdna3.1+
(wherein, the sequence accession number of basic-pcdna3.1+ is lq014872, and the sequence accession number of cdx2 is gu017420) transfection
Enter in ipec-j2 cell line, stably express the cell strain of the ipec-j2 of cdx2 with suitable g418 concentration screening, fixed with fluorescence
The ipec-j2 cell strain of cdx2 is stably expressed in the method identification of amount pcr and western blotting, specifically comprises the following steps that
(1) wild type ipec-j2 cell is inoculated in 6 orifice plates, inoculum density is 1 × 106Cells/ml, inoculation volume is 2
Ml/ hole, is placed in containing 5% co2Incubator in cultivate, cultivation temperature be 37 DEG C.
(2) cultivate 3～4 days, to 80%～90% fusion, complete medium is cell growth: dmem+10% fbs.
(3) prepare transfection liquid:
Use opti-mem®I low blood serum medium dilution plasmid dna (basic-pcdna3.1+ and cdx2-pcdna3.1+),
Mix homogeneously.
Dilute lipofectamin 3000, incubated at room 5 minutes with the low blood serum medium of opti-mem i.
3. willWithThe plasmid dna being diluted and lipofectamin3000 mix homogeneously, incubated at room 20 minutes, system
Become transfection liquid.
(4) add the transfection liquid of 500 l step (3) gained in the cell of every hole, gently shake up, continue to be placed in incubator
Culture.
(5) complete medium that culture more renewed after 5 hours.
(6), after culture transfects 24 hours, with 0.25% trypsin-edta, cell dissociation is got off, with fresh training completely
It is inoculated in 6 orifice plates after foster base dilution, be initially added within second day 400 g/ml g418 and screened, change a not good liquor within 3 days, treat the moon
Property compared with control cells all after death, g418 concentration is changed to 200 g/ml, cell clone is selected in culture after about 14 days, expands training
Support.
(7) obtain basic-ipec-j2 and cdx2-ipec-j2 cell strain, both morphologically no significant difference (Fig. 1).
Embodiment 2 real time fluorescent quantitative pcr detects the mistake of the ipec-j2 cell strain of overexpression cdx2 that embodiment 1 obtains
Expression
First, the extracting of total rna:
(1) collect basic-ipec-j2 the and cdx2-ipec-j2 cell sample that embodiment 1 prepares respectively, add 1 ml
Trizol lysate (invitrogen, carlsbad, usa), room temperature stands 5min；(2) use eppendorf 5810r centrifuge
12000 rpm, 4 DEG C of centrifugation 15 min；(3) collect supernatant, add 0.2 ml chloroform, mix, room temperature stands 5 min；(4) use
Eppendorf 5810r centrifuge 12000 rpm, 4 DEG C of centrifugation 15 min；(5) take supernatant, add isopyknic isopropanol,
Mix, -80 DEG C of standing 30 min；(6) eppendorf 5810r centrifuge 12000 rpm, 4 DEG C of centrifugation 15 min are used；(7) abandon
Supernatant, adds the ethanol solution that 1 ml concentration is 75 % toward in precipitate, and under the conditions of 4 DEG C, 12000rpm is centrifuged 10 min；(8)
Repeat step (7)；(9) abandon supernatant, put the precipitate in drying at room temperature, be changed into transparence to white precipitate；(10) add in right amount
Depc water dissolution after obtain total rna solution of basic-ipec-j2 and cdx2-ipec-j2 cell.
2nd, in total rna dna digestion: (1) takes total rna sample of 12 μ g gained, adds 0.25 μ l rnase
Inhibitor(takara, shiga, japan), 0.5 μ l dnase i(takara, shiga, japan), 2 μ l 10 ×
Dnase i buffer(takara, shiga, japan), add depc water to cumulative volume 20 μ l, mix；(2) 37 DEG C of reactions
30 min, 65 DEG C of reaction 5 min.
3rd, cdna synthesis: (1) takes the postdigestive rna of 2 μ g, adds 30 pmol random primer n10(nnnnnnnnnn；n=
A, t, c, g), plus depc water is to 15 μ l, 70 DEG C of degeneration 5 min, 4 DEG C of cooling 5 min；(2) add 5 μ l m-mlv 5 ×
Buffer(promega, madison, wi, usa), 2.5 μ l dntp mixture(10 mmol/l, Beijing SBS Genetech gene skill
Art company limited, Beijing), 0.25 μ l rnase inhibitor and 1.25 μ l depc water；(3) 37 DEG C of insulation 60 min；
80 DEG C of inactivations 5 min, 12 DEG C of 10 min；(4) obtain basic-ipec-j2 and cdx2-ipce-j2 cell cdna sample.
4th, real time fluorescent quantitative pcr:(1) add 2 μ l to obtain in the middle of special 96 orifice plates of fluorescent quantitation pcr respectively
Basci-ipec-j2 and cdx2-ipce-j2 cell cdna, 10 pmol cdx2 primers (forward primer: 5 '-
gtcgctacatcaccattcgg-3′；Downstream primer r:5 '-gattttcctctccttcgctct-3 '), 9 μ l sybr
Green real-time pcr master mix(toyobo, tokyo, japan) and 9 μ l depc water；(2) covered with sealed membrane
Cover on pcr plate, compression is it is ensured that each hole is airtight；(3) it is placed in mx3005p type real time fluorescent quantitative pcr instrument (agilent
Technologies, santa clara, usa) in: (95 DEG C of 60 s), (95 DEG C of 15 s；58 ℃ 40 s；72 ℃
40 s) × 35 cycles, (95 DEG C of 60 s；58 ℃ 15 s；72 DEG C 30 s)；(4) with β-actin as internal standard gene,
Cdx2 relative expression quantity=2[ct (β-actin)−ct (cdx2)].
Result display cdx2-ipec-j2 cell cdx2 mrna expression is significantly higher than basic-ipec-j2 cell (p ＜
0.01) (Fig. 2).
Embodiment 3 western blotting detects the ipec-j2 cell strain of overexpression cdx2 that embodiment 1 obtains
Overexpression situation
First, cell protein extracts: (1) collects basic-ipec-j2 and cdx2-ipec-j2 cell sample, adds ripa cracking
Liquid；(2) 4 DEG C of standing 30 min；(3) 4 DEG C of centrifugation 5 min of eppendorf 5810r centrifuge 12000 rpm are used；(4) collect
Supernatant, as total protein of cell sample.
2nd, according to sds-page gel reagents box (green skies biotechnology research institute) operation instruction, preparation concentration is 10%
Separation gel and concentration glue that concentration is 5%, loading runs electrophoresis.
3rd, transferring film: the gel after electrophoresis is terminated by (1) takes out from glass plate, excise redundance, remaining containing mesh
Albumen separation gel, be soaked in transferring film buffer (in 1 l deionized water contain 3.02 g tris alkali, 14.4 g glycine and
200 ml methanol) in；(2) in transferring film folder, sponge, whatman 3# filter paper, gel, pvdf film, whatman are installed in order
3# filter paper, sponge；(3) transferring film is folded up in transfer groove, adds 4 DEG C of transferring film buffer, 100 v constant voltages shift 70 min,
Destination protein is made to be transferred in pvdf film.
4th, pvdf film is placed in 5 % bsa, room temperature closes 2 h.
5th, press 1:5000 dilution cdx2 protein antibodies, pvdf film processed for step 4 is placed in cdx2 protein antibodies
In, 4 DEG C of overnight incubation, incubation is washed 6 times with tbst after terminating, 5 min every time.
6th, the two anti-1:10000 that press dilute, 4 DEG C of incubation 2 h, and pvdf film is taken out after terminating, washed with tbst 6 times by incubation,
5 min every time.
7th, prepare ecl chemical luminescence for liquid to specifications, pvdf film is soaked in luminescent solution, is placed in fluorchem m
Apparatus gel imaging instrument exposes, and obtains stripe information.
8th, use image j software statistics band gray value.
Result display cdx2-ipec-j2 cell cdx2 expressing quantity is significantly higher than basic-ipec-j2 cell (p ＜
0.01) (Fig. 3).
Embodiment 4 prepares cdx2 Conditioned immunolresponse
Complete medium used by the present embodiment is: adds 10% fbs, 1% penicillin/strepto- in advanced dmem/f12
Element, 2 mmol/l l- L-Glutamine and 10 mmol/l hepes.
(1) cdx2-ipec-j2 cell is inoculated in 75 cm2Tissue Culture Flask in (add culture medium: advanced
10% fbs, 0.4 mg/ml g418,2 mmol/l L-Glutamine and 10 mmol/l hepes are added in dmem/f12), it is placed in
Cultivate in CO2 gas incubator；Change a not good liquor within 2 days.
(2) when cell fusion is to 80%, remove culture medium, with pbs rinse 2 times, add 4 ml 0.25% trypsin-
Edta(gibco, carlsbad, ca), 37 DEG C of incubation 10 min, add 4 ml culture medium to terminate trypsin-edta digestion.
(3) eppendorf 5810r centrifuge 1000 rpm, 4 DEG C of centrifugation 3 min are used to collect cell.
(4) use complete medium resuspended cdx2-ipec-j2 cell.
(5) it is inoculated in 75 cm by 1:102In Tissue Culture Flask, it is placed in culture in CO2 gas incubator.
(6) after cultivating 4 days, collect supernatant, with 0.24 μm of membrane filtration, collect under the conditions of filtrate 1 is placed in 4 DEG C and preserve.
(7) add 10 ml complete mediums to continue culture toward in culture bottle, collect supernatant again after 2 days, use 0.24 μ
M membrane filtration, collects filtrate 2.
(8) by filtrate 1 and filtrate 2 according to 1:1 volume mixture, as cdx2 Conditioned immunolresponse.
(9) preserve under the conditions of cdx2 Conditioned immunolresponse being placed in 4 DEG C.
Embodiment 5 pig small intestine crypts single cell culture
(1) take suckling pig small intestinal, separate its crypts unit, add appropriate Digestive system (Digestive system: advanced dmem/
1% n2,2% b27,1% penicillin/streptomycin, 1 mmol/l n- acetyl-cysteine, 2 mmol/l l- paddy are added in f12
Glutamine, 10 mmol/l hepes, 10 μm of ol/l y27632 and 0.3 u/ml dispase i), 120 37 DEG C of rpm water-baths
30 min.
(2) volume according to Digestive system adds 10% fbs and 50 μ g/ml dnase i to terminate digesting, 1000 rpm, 4 DEG C
It is centrifuged 3 min, remove supernatant, (in advanced dmem/f12, add 1% n2,2% b27,1% penicillin/strepto- with storing liquid
Element, 1 mmol/l n- acetyl-cysteine, 2 mmol/l l- L-Glutamine, 10 mmol/l hepes and 10 μm of ol/l
Y27632) re-suspended cell precipitation, is filtered with 200 mesh, 400 mesh and 500 mesh cell sieves successively, obtains pig small intestine crypts unicellular.
(3) calculate the single celled density of crypts with counting method of blood cell, be blended in matrigel according to 50 cells/ml,
It is inoculated in 48 orifice plates according to 25 μ l/well, 37 DEG C of 5% co2Cultivating 25 min makes matrigel solidify.
(4) add basal medium (basal medium: add in advanced dmem/f12 according to 250 μ l/well
100 ng/ml wnt3a, 1% n2,2% b27,1 mmol/l n- acetyl-cysteine, 2 mmol/l l- L-Glutamine, 10
μm ol/l sb202190,0.5 μm of ol/l ly2157299,50 ng/ml egf, 100 ng/ml noggin and 500 ng/ml
R-spondin 1) to be cultivated, it is unicellular that Intestinum Sus domestica road crypts is cultivated in this culture medium success in vitro.
Embodiment 6
Experimental technique with embodiment 5, unique unlike, the basal medium used by the present embodiment is: advanced dmem/
Add in f12 100 ng/ml wnt3a, 1%n2,2%b27,1% penicillin/streptomycin, 1 mmol/l n- acetyl-cysteine,
2 mmol/l l- L-Glutamine, 10 mmol/l hepes, 10 mmol/l nicotine, 10 μm of ol/l sb202190,0.5 μ
Mol/l ly2157299,50 ng/ml egf, 100 ng/ml noggin and 500 ng/ml r-spondin 1, cultivate 8d,
Result such as Fig. 4.
Embodiment 7
Experimental technique with embodiment 6, unique unlike, the basal medium used by the present embodiment is: advanced dmem/
200 ng/ml wnt3a, 2% n2,1% b27,1% penicillin/streptomycin, 0.5 mmol/l n- acetyl-half Guang is added in f12
Propylhomoserin, 1 mmol/l l- L-Glutamine, 5 mmol/l hepes, 5 mmol/l nicotine, 5 μm of ol/l sb202190,1 μ
Mol/l ly2157299,100 ng/ml egf, 200 ng/ml noggin and 1000 ng/ml r-spondin 1.
Embodiment 8 pig small intestine crypts single cell culture
The basal medium of embodiment 6, with embodiment 6, is uniquely except for the difference that replaced with Optimal Medium 1 by experimental technique, described
The consisting of of Optimal Medium 1: cdx2 Conditioned immunolresponse described in embodiment 4,100 ng/ml wnt3a, 1% n2,2% b27,
1% penicillin/streptomycin, 1 mmol/l n- acetyl-cysteine, 2 mmol/l l- L-Glutamine, 10 mmol/l hepes,
10 mmol/l nicotine, 10 μm of ol/l sb202190,0.5 μm of ol/l ly2157299,50 ng/ml egf, 100 ng/ml
Noggin and 500 ng/ml r-spondin 1, wherein volume fraction in Optimal Medium 1 for the cdx2 Conditioned immunolresponse is
15～25 v/v%.
Result shows, with respect to basal medium described in embodiment 6, Optimal Medium 1 promotes Intestinum Sus domestica road pit cell body
The effect of outer amplification is more preferably (Fig. 5).
Embodiment 9 pig small intestine crypts single cell culture
The basal medium of embodiment 6, with embodiment 6, is uniquely except for the difference that replaced with Optimal Medium 2 by experimental technique, described
The consisting of of Optimal Medium 2: cdx2 Conditioned immunolresponse described in embodiment 4,100 ng/ml wnt3a, 1% n2,2% b27,
1% penicillin/streptomycin, 1 mmol/l n- acetyl-cysteine, 2 mmol/l l- L-Glutamine, 10 mmol/l hepes,
10 mmol/l nicotine, 10 μm of ol/l sb202190,0.5 μm of ol/l ly2157299,50 ng/ml egf, 100 ng/ml
Noggin and 500 ng/ml r-spondin 1, wherein volume fraction in Optimal Medium 2 for the cdx2 Conditioned immunolresponse is
40～50 v/v%.
Result shows, with respect to basal medium described in embodiment 6, Optimal Medium 2 promotes Intestinum Sus domestica road pit cell body
The effect of outer amplification is more preferably (Fig. 6).
Embodiment 10 pig small intestine crypts single cell culture
With embodiment 6, after unique except for the difference that step (4) addition culture medium, cultural method arranges 3 experimental grouies to experimental technique,
Experimental group 1: culture whole (8d) uses basal medium described in embodiment 6；Experimental group 2: culture whole (8d) uses embodiment 9
Described Optimal Medium 2；Experimental group 3: culture early stage (1～5d) use basal medium described in embodiment 6, late stage of culture (6～
8d) use Optimal Medium 2 described in embodiment 9.
Result shows, the cultural method of experimental group 3 cultivates effect optimum (Fig. 7) of Intestinum Sus domestica road pit cell.
Comparative example 1
Experimental technique with embodiment 6, unique unlike, there is no 100 ng/ml wnt3a, 50 ng/ml in basal medium
Egf, 100 ng/ml noggin500 ng/ml r-spondin 1, cell such as Fig. 8 after cultivating 8 days.
Comparative example 2
Experimental technique with embodiment 6, unique unlike, there is no 10 μm of ol/l sb202190 and 0.5 μ in basal medium
Mol/l ly2157299, cell such as Fig. 9 after cultivating 8 days.
Claims (6)
1. a kind of culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro is it is characterised in that the consisting of of described culture medium:
In advanced dmem/f12 culture fluid contain 100～200 ng/ml wnt3a, 1～2% n2,1～2% b27,0.5～1
Mmol/l n- acetyl-cysteine, 1～2 mmol/l l- L-Glutamine, 5～10 μm of ol/l sb202190,0.5～1 μ
Mol/l ly2157299,50～100 ng/ml egf, 100～200 ng/ml noggin and 500～1000 ng/ml r-
spondin 1.
2. the culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro according to claim 1 is it is characterised in that described
Promote the consisting of of culture medium of Intestinum Sus domestica road pit cell amplification in vitro: cdx2 conditioned medium, 100～200 ng/ml
Wnt3a, 1～2% n2,1～2% b27,0.5～1 mmol/l n- acetyl-cysteine, 1～2 mmol/l l- glutamy
Amine, 5～10 μm of ol/l sb202190,0.5～1 μm of ol/l ly2157299,50～100 ng/ml egf, 100～200
Ng/ml noggin and 500～1000 ng/ml r-spondin 1；Described cdx2 conditioned medium is in described promotion Intestinum Sus domestica road
Volume fraction in the culture medium of pit cell amplification in vitro is 15～50 v/v%；Described cdx2 conditioned medium is by following step
Rapid acquisition: (1) will be a number of, after the cell containing cdx2 gene is cultivated 3.5～4 days in complete medium, in collection
Clear liquid, obtains filtrate 1 after filtration；(2) continue the cell containing cdx2 gene for the culture, after cultivating 2～3 days, collect supernatant again,
Filter to get filtrate 2, filtrate 1 and filtrate 2 are obtained cdx2 Conditioned immunolresponse by 1:1～0.8:1 volume mixture.
3. the culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro according to claim 2 is it is characterised in that described
Complete medium be in advanced dmem/f12 culture fluid contain 5～10% fbs, 0.5～1% penicillin/streptomycin, 1～2
Mmol/l l- L-Glutamine and 5～10 mmol/l hepes.
4. the culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro according to claim 1 is it is characterised in that described
Culture medium also contains 1～2% penicillin/streptomycin or/and 5～10 mmol/l hepes or/and 5～10 mmol/l nicotine.
5. a kind of method promoting Intestinum Sus domestica road pit cell amplification in vitro is it is characterised in that add in Intestinum Sus domestica road crypts is unicellular
Enter the culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro described in any one of claim 1 to 4 to be cultivated.
6. a kind of method promoting Intestinum Sus domestica road pit cell amplification in vitro is it is characterised in that cultivate early stage, in Intestinum Sus domestica road crypts list
The culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro described in claim 1 is added to be cultivated in cell；Late stage of culture
It is replaced by the culture medium promoting Intestinum Sus domestica road pit cell amplification in vitro described in claim 2 to be cultivated.
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| CN108085296A (en) * | 2018-01-29 | 2018-05-29 | 清华大学 | Culture medium and application thereof |
| CN108085296B (en) * | 2018-01-29 | 2021-04-13 | 清华大学 | Intestinal epithelium organoid growth factor-free culture method |
| CN109161516A (en) * | 2018-09-13 | 2019-01-08 | 华中农业大学 | A kind of method of chitling road crypts separation and the culture of 3D organoid |
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| CN114404573A (en) * | 2021-12-24 | 2022-04-29 | 华南农业大学 | Application of Wnt3a protein in promoting animal growth |
| CN114717182A (en) * | 2022-03-17 | 2022-07-08 | 中国科学院亚热带农业生态研究所 | Molecular culture medium for 3D culture of goat colon organoid |
| CN115340960A (en) * | 2022-03-18 | 2022-11-15 | 广东省农业科学院动物科学研究所 | Method for constructing co-culture system of porcine intestinal organoid and enterotoxigenic escherichia coli or macrophage |
| CN115340960B (en) * | 2022-03-18 | 2024-06-18 | 广东省农业科学院动物科学研究所 | Construction method of pig intestinal organoid and enterotoxigenic escherichia coli or macrophage co-culture system |
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