CN102329770A - Improved manufacturing method for mouse embryonic bodies - Google Patents

Abstract

The invention discloses an improved mouse embryoid body production method, which comprises the steps of culturing mouse ES cells, preparation of the embryoid body, suspension culture of the embryoid body and adherent culture of the embryoid body. The present invention uses trypsin-containing cell digestion solution to digest mouse ES cells into cell colonies, so that the cells in these cell colonies are completely aggregated together and separated from the feeder cells, while the feeder cells are digested into individual cells, so that high-purity, high-quality mouse ES cell embryoid bodies can be directly produced.

Description

Improved mouse embryoid body making method

[technical field]

The present invention relates to animal embryoid body making method, relate in particular to a kind of improved mouse embryoid body making method.

[background technology]

Be accompanied by the growth at age; Particularly in modern times the society along with the progress of society and the ratio of brainwork increase; People's physique descends rapidly; Human old degenerative disorders appears multiple and occurred frequently, like the pancreatic cell of excreting insulin the Parkinson's disease that the neurocyte degeneration of the mellitus that cause, secretory nerve mediator Dopamine HCL causes etc. of degenerating.In addition, some familial inheritance property diseases, the familial inheritance property disease that causes owing to the sudden change of genetic information is like Mediterranean Sea formula anaemia, hemophilia, albinism etc.Most effectual way for these diseases can thoroughly be cured is utilized embryonic stem cell (Embryonic stem cell exactly; The ES cell) induces differentiation technique; Mouse is as a kind of important experimental animals model, and the Study on Differentiation of inducing of ES cells will be established solid basis for the successful implementation of human regenerative medicine.The ES cell is a kind of multipotent stem cells colony that derives from the blastaea inner cell mass, has stronger regenerative power and plasticity-, under the vitro culture envrionment conditions, can carry out genetic modification operations such as foreign gene importing, gene knockout or genetic modification efficiently.The ES cell can be differentiated to form all types of composition cells of body under certain induced environment condition, comprises female ovocyte and male sperm.The ES cell will be established important basis for healthy reproduction of production, rearing new variety, the conservation of wildlife and the people of transgenic animal etc. to the differentiation of ovocyte and sperm, has extremely strong using value and realistic meaning.The ES cell of process genetic manipulation is at the external evoked specific cells that is divided into; Can therapeutically effective genetic property disease; Through to the external evoked research that is divided into specific cells of ES cells, for the fast development of human regenerative medicine and the biotherapy of human diseases are established solid basis.The successful making of embryoid body is platform and the basis of ES cell to the efficient differentiation of specific cells.

In cell dissociation goes down to posterity cultivation; The cell dissociation buffer that contains 2.5mg/ml pancreatin-0.2mg/ml EDTA is conventional commercialization reagent; Use the cell dissociation buffer that contains 2.5mg/ml pancreatin-0.2mg/ml EDTA to be digested to individual cells to the ES cell together with feeder layer cells in the making of conventional embryoid body; Adopt reversal of the natural order of things enchylema hanging drop to these blended cell colonys; The cell that mixes in the enchylema hanging drop forms cell mass jointly, comprises more feeder layer cells in the embryoid body cell mass that forms like this, because feeder layer cells is mingled in the embryoid body; Greatly reduced the quality of embryoid body; Can cause the differentiation efficiency in downstream to reduce and the differentiation downgrade, thereby have a strong impact on the differentiation efficiency of multipotent stem cells, weaken the clinical of ES cell and the using value and the meaning of producing to sexual cell sperm or ovum and other cell types.

[summary of the invention]

The technical problem that the present invention will solve provides a kind of improved mouse embryoid body making method, and it is made simply, effect is obvious, the embryoid body reliable in quality of making.

In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is that improved mouse embryoid body making method comprises the steps:

(1) cultivation of ES cells

Getting 12.5d mouse fetal inoblast cultivates with high sugared DMEM nutrient solution; After treating cell confluent culture ware; Cultivate with the cell dissociation buffer had digestive transfer culture that contains 2.5mg/ml pancreatin-0.2mg/ml EDTA; Get the MTC effect 2.5h of the fetal fibroblast of confluent culture ware with 10 μ g/ml; With in incubator, cultivating on the petridish that is seeded to the processing of 0.1% gelatin after the cell dissociation buffer digestion that contains 2.5mg/ml pancreatin-0.2mg/ml EDTA, the cell of getting the even confluent culture ware of inoculation back 2d is as feeder layer; ES cells R1 clone is commercialization clone, gets the 26th generation ES cells R1 clone and is inoculated on the mouse fetal inoblast feeder layer, adds the high sugared DMEM nutrient solution that contains 1000U/ml LIF and cultivates;

(2) making of embryoid body

Getting ES cells washs with PBS; Add the cell dissociation buffer that contains the 2.5mg/ml pancreatin and be positioned over incubator digestion 3min, gently shake the beating petridish, collect the ox iPS cell colony that comes off with have gentle hands; Centrifugal; The PBS washing, centrifugal again back is resuspended with the high sugared DMEM nutrient solution of removing 1000U/ml LIF, and inoculating cell is cultivated to the low Micro-Organism Culture Dish incubator that attaches;

(3) suspension culture of embryoid body

The 2d of inoculation culture; From the low Micro-Organism Culture Dish that attaches, cell culture fluid is transferred in the centrifuge tube together with embryoid body; Room temperature is static; After treating that embryoid body natural sedimentation that cell colony forms is at the bottom of the centrifuge tube, shift embryoid body and be inoculated in the Micro-Organism Culture Dish of new low attaching, add the high sugared DMEM nutrient solution of removing 1000U/ml LIF and in incubator, cultivate;

(4) adherent culture of embryoid body

Embryoid body is cultivated a week in the low Micro-Organism Culture Dish that attaches after; From the low Micro-Organism Culture Dish that attaches, cell culture fluid is transferred in the centrifuge tube together with embryoid body; Room temperature is static, treat that embryoid body natural sedimentation that cell colony forms is at the bottom of the centrifuge tube after, shift embryoid body and be inoculated in the ordinary cells petridish that gelatin encapsulates; Add the high sugared DMEM nutrient solution of removing 1000U/ml LIF and in incubator, cultivate, promptly can be used for external triploblastica differentiation capability after the week and detect.

The invention has the beneficial effects as follows:

The cell dissociation buffer that employing contains the 2.5mg/ml pancreatin is cell colony with ES cells digestion, makes that cell intactly flocks together in these cell colonies, and separate from feeder layer cells that feeder layer cells then is digested individual cells.Therefore, adopt the ES cell of the cell dissociation buffer digestion that contains the 2.5mg/ml pancreatin can directly make high purity, high quality ES cells embryoid body.Thereby the cell that is divided into the needed other types of body for the ES cells efficient induction is laid a good foundation.

[description of drawings]

Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation.

Fig. 1 is the growthhabit that is grown in ES cells on the feeder layer under the state of nature;

Fig. 2 is the growthhabit that the suspension culture ES cells forms embryoid body;

[embodiment]

One, material and method

(1) cultivation of ES cells

Getting 12.5d mouse fetal inoblast cultivates with high sugared DMEM nutrient solution; After treating cell confluent culture ware; Cultivate with the cell dissociation buffer had digestive transfer culture that contains 2.5mg/ml pancreatin-0.2mg/ml EDTA; Get the MTC effect 2.5h of the fetal fibroblast of confluent culture ware with 10 μ g/ml; With in incubator, cultivating on the petridish that is seeded to the processing of 0.1% gelatin after the cell dissociation buffer digestion that contains 2.5mg/ml pancreatin-0.2mg/ml EDTA, the cell of getting the even confluent culture ware of inoculation back 2d is as feeder layer.ES cells R1 clone is commercialization clone, gets the 26th generation ES cells R1 clone and is inoculated on the mouse fetal inoblast feeder layer, adds the high sugared DMEM nutrient solution that contains 1000U/ml LIF and cultivates.

(2) immunofluorescence and alkaline phosphatase staining detect

ES cells is fixed through the Paraformaldehyde 96 room temperature, and the PBS solution of Triton X-100 is penetrating, after the PBS solution sealing of BSA; Be added into respectively after the one anti-Oct4 of being, Nanog, SSEA-1 diluted by 1: 200 and hatch altogether on the cell; PBS washing, two of interpolation red fluorescence mark resist by 1: 200 dilution back room temperature lucifuge hatches the PBS washing altogether; The DAPI nucleus dyeing of 1 μ g/ml, fluoroscope is observed down.ES cells is fixed through Paraformaldehyde 96, adds the SEAP dye liquor and dyes, and is colored as reddish-brown or coffee-like particle is positive with endochylema.

(3) making of embryoid body

Getting ES cells washs with PBS; Add the cell dissociation buffer that contains the 2.5mg/ml pancreatin and be positioned over incubator digestion 3min, gently shake the beating petridish, collect the ox iPS cell colony that comes off with have gentle hands; Centrifugal; The PBS washing, centrifugal again back is resuspended with the high sugared DMEM nutrient solution of removing 1000U/ml LIF, and inoculating cell is cultivated in the incubator in the low Micro-Organism Culture Dish that attaches.

(4) suspension culture of embryoid body

The 2d of inoculation culture; From the low Micro-Organism Culture Dish that attaches, cell culture fluid is transferred in the centrifuge tube together with embryoid body; Room temperature is static; After treating that embryoid body natural sedimentation that cell colony forms is at the bottom of the centrifuge tube, shift embryoid body and be inoculated in the Micro-Organism Culture Dish of new low attaching, add the high sugared DMEM nutrient solution of removing 1000U/ml LIF and in incubator, cultivate.

(5) adherent culture of embryoid body

Embryoid body is cultivated a week in the low Micro-Organism Culture Dish that attaches after; From the low Micro-Organism Culture Dish that attaches, cell culture fluid is transferred in the centrifuge tube together with embryoid body; Room temperature is static, treat that embryoid body natural sedimentation that cell colony forms is at the bottom of the centrifuge tube after, shift embryoid body and be inoculated in the ordinary cells petridish that gelatin encapsulates; Add the high sugared DMEM nutrient solution of removing 1000U/ml LIF and in incubator, cultivate, be used for external triploblastica differentiation capability after the week and detect.

(6) vitro differentiation of embryoid body detects

ES cells vitro differentiation embryoid body is fixed through the Paraformaldehyde 96 room temperature, and the PBS solution of Triton X-100 is penetrating, after the PBS solution sealing of BSA; The one anti-α-actinin (Sarcomeric) of being, α-Fetoprotein (AFP), Neurofilament 200, resist by being added into respectively after the dilution in 1: 200 hatches the PBS washing altogether on the cell; Two of red fluorescence mark resists by 1: 200 dilution back room temperature lucifuge hatches altogether; The PBS washing, the DAPI nucleus dyeing of 1 μ g/mL, fluoroscope is observed down.

Two, result

(1) growth conditions of ES cells

ES cells is on 12.5d fetal fibroblast feeder layer, and form is more consistent, and cell colony is nest like (Fig. 1); Colony is obvious to upper process; Fast growth, under the situation by 1: 3 had digestive transfer culture, the ES cell is separated from cell colony with the cell dissociation buffer digestion back that contains 2.5mg/ml pancreatin-0.2mg/ml EDTA; That cell is is single, volume is less, and the ES cell colony 2d that on feeder layer, grows goes down to posterity once.After the effect of SEAP dye liquor, the alkaline phosphatase staining of ES cells colony is strong positive, and cell colony is red-brown or red-purple.ES cells high level expression stem cell labeling albumen Oct4, Nanog and SSEA-1.The result shows that ES cells keeps good not differential growth state.

(2) formation of ES cells embryoid body

ES cells is after the cell dissociation buffer digestion that contains the 2.5mg/ml pancreatin; The ES cell colony is kept perfectly, is cell clone, and in the petridish of low adhesive power, cell clone directly forms embryoid body; The embryoid body form that forms is a spheroidal, smooth surface bright (Fig. 2).The ES cell colony is inoculation back 1d on the Micro-Organism Culture Dish of low adhesive power; The feeder layer cells that digests from petridish can adherent growth on the Micro-Organism Culture Dish of low adhesive power; Adherent feeder layer cells is removed when the petridish that the ES cells embryoid body more renews, and makes the ES cell embryoid body that forms and has higher cell purity.Be accompanied by the prolongation of ES cell embryoid body incubation time, embryoid body is formed iuntercellular and is connected the comparatively densification that progressively becomes, and is prone to mutual adhesion between embryoid body and forms bigger embryoid body colony, and the color of embryoid body progressively from light to dark.

(3) triploblastica of ES cells embryoid body differentiation

The ES cells embryoid body through after the suspension culture in the ordinary cells petridish that gelatin encapsulates adherent growth; Adherent embryoid body presents spontaneous differentiation; Detect demonstration through immunocytochemistry; Embryoid body with ES cells making of versatility can be differentiated to form mesoderm myocardial cell, the entoderm liver cell of express alpha-Fetoprotein (AFP), the expression Neurofilament ectoderm neurocyte of express alpha-actinin (Sarcomeric) efficiently to the composition cytodifferentiation of three germinal layers of body.

Present embodiment is through this simple ES cells embryoid body making method; ES cell embryoid body is formed cell at external three germinal layers that can be divided into body, thereby establishes solid basis for utilizing ES cells embryoid body manufacturing technology to induce formation body all types to form cell and even sexual cell sperm and ovum.

Claims (1)

1. An improved mouse embryoid body production method, comprising the steps of: (1) Culture of mouse ES cells Fetal fibroblasts from 12.5-day-old mice were cultured with high-sugar DMEM medium. After the cells covered the culture dish, they were digested and subcultured with cell digestion solution containing 2.5mg/ml trypsin-0.2mg/ml EDTA. The fetal fibroblasts in the full culture dish were treated with 10μg/ml mitomycin for 2.5h, digested with the cell digestion solution containing 2.5mg/ml trypsin-0.2mg/ml EDTA, and inoculated into the culture dish treated with 0.1% gelatin Cultured in the incubator above, and the cells that evenly covered the culture dish on the 2nd day after inoculation were used as the feeder layer; the mouse ES cell R1 cell line was a commercial cell line, and the 26th generation mouse ES cell R1 cell line was inoculated On the feeder layer of mouse fetal fibroblasts, add high-sugar DMEM medium containing 1000U/ml LIF for cultivation; (2) Preparation of embryoid bodies Take mouse ES cells and wash them with PBS, add cell digestion solution containing 2.5mg/ml trypsin, place them in the incubator for digestion for 3 minutes, gently shake and pat the culture dish with your hands, collect the fallen bovine iPS cell colonies, centrifuge, wash with PBS, and then After centrifugation, resuspend with high-sugar DMEM culture medium with 1000U/ml LIF removed, and inoculate the cells into a low-attachment bacterial culture dish for culture in an incubator; (3) Suspension culture of embryoid bodies On the second day of inoculation culture, transfer the cell culture medium together with the embryoid bodies from the low-attachment bacterial culture dish to a centrifuge tube, and let it stand at room temperature. After the embryoid bodies formed by cell colonies naturally settle to the bottom of the centrifuge tube, transfer The embryoid body was inoculated into a new low-attachment bacterial culture dish, and the high-sugar DMEM culture solution with 1000U/ml LIF removed was added to culture in the incubator; (4) Adhesive culture of embryoid bodies After the embryoid bodies are cultured in a low-attachment bacterial culture dish for one week, transfer the cell culture medium together with the embryoid bodies from the low-attachment bacterial culture dish to a centrifuge tube, and let it stand at room temperature until the embryoid bodies formed by cell colonies After the body naturally precipitated to the bottom of the centrifuge tube, the embryoid body was transferred and inoculated into a gelatin-coated ordinary cell culture dish, and the high-sugar DMEM culture solution with 1000U/ml LIF removed was added to culture in the incubator, and it could be used in vitro after one week Three germ layer differentiation ability detection.

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