CN203411544U

CN203411544U - Aorta three-dimensional culture single cell separator

Info

Publication number: CN203411544U
Application number: CN201320241787.1U
Authority: CN
Inventors: 颜凤; 蒋凡
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-04-25
Filing date: 2013-04-25
Publication date: 2014-01-29
Anticipated expiration: 2023-04-25

Abstract

The utility model relates to a device for animal tissue culture in vitro and cell culture, namely an aorta three-dimensional culture single cell separator. The single cell separator comprises two glass slides (I and II), wherein the glass slide I is a common glass slide, and a layer of rat tail collagen is attached to the upper surface of the glass slide I; a hole is formed in the glass slide II, and a layer of Dow Corning organosilicon pouring sealant is attached to the lower surface of the glass slide II; the processed surfaces of the two glass slides are bonded together through double-faced adhesive tapes; an ice-bathed liquid matrigel is poured to the hole of the glass slide II and packed between the two glass slides. The device has the advantages as follows: an angiogenesis condition is simulated really; the operation is relatively simple; observation is easy. The device can further be used for single cell study and isolated culture, and a single cell growing on the glass slides can be seen clearly and observed easily. The device is mainly used for angiogenesis single cell culture, and isolation and study on single cells.

Description

Aorta dimensional culture single cell separator

Technical field

The utility model relates to a kind of device of a kind of animal tissues vitro culture and cell cultures.

Background technology

Angiogenesis (angiogenesis) is to germinate and generate the process of neovascularity from existing blood vessel.Angiogenesis is found in physiological status, as the normal growth of embryo's formation and tissue.In addition, angiogenesis is common in some pathologic condition, as tumour, wound healing etc.Angiogenesis has important value in the research of tumour, studies confirm that at present new vessel not only can increase knurl body, and makes tumour more easily occur to shift and diffusion, finally causes serious consequence.The angiogenesis of research tumour contributes to understand that malignant tumour occurs, development, invasion and attack and the biological characteristics that shifts, and has important theory significance and clinical value to illustrating mechanism, estimating prognosis and carrying out antiangiogenic therapy etc.

Set up both at home and abroad the model of many research new vesseles, external model is based on endotheliocyte, on supported matrix, to cultivate the principle foundation that can form luminal structure, as cell or tissue culture model.Body inner model is to study in animal body angiogenesis, as corneal vascularization model, and chick chorioallantoic membrane angiogenic growth model and polymkeric substance planting model.

Corneal vascularization model is that somatomedin is expelled on cornea, or is added in the pouch that cornea upper cut forms, by slit lamp microscope observation sinuys venosus sclerae plexus vasculosus to the situation of growing in cornea.Corneal test operation technique difficulty is large, and operation is also unavoidable afterwards exists inflammatory reaction, and the blood vessel generating is irregular, is difficult to quantitatively.

Chick chorioallantoic membrane angiogenic growth model (CAM) is that medicine is added to carrier as on gelfoam, methylcellulose gum, cellulose mixture filter membrane or filter paper, is placed on and windows on the chorioallantoic membrane exposing, and observes angiogenic growth situation on chorioallantoic membrane after three days.CAM model is applicable to large-scale medicine screening, but the blood vessel network of CAM itself makes newly-generated blood vessel be difficult to identification, and starts there is inflammatory reaction in 7 to 8 days, has hindered the resolution of new vessel.

Polymkeric substance planting model, conventional matrigel or sponge.Matrigel method is that matrigel is expelled in mouse or rat body, and temperature raises and makes matrigel solidify to form embolus.After 7 days, with histochemical method or Immunohistochemical Method, detect the blood vessel situation of embolus of growing into, or detect content of hemoglobin in embolus.The shortcoming of this method is that matrigel is expensive, analyzes more consuming time.

In external model cell culture model be by endothelial cell seeding in having overlay the culture dish of collagen or scleroproein, matrigel, observe the capillary vessel spline structure that endotheliocyte forms in the plane.This model manipulation is simple, can show cell by dormancy the process to blastogenesis, but because cell does not grow from existing blood vessel, can not simulate real angiogenesis completely.

Organize models is embedded in matrix as cultivated in the mixture of the scleroproein of collagen gel, plasma clot, purifying, matrigel or these several albumen by explant as aortic annulus, observes the endotheliocyte growing from inner membrance and to surrounding, form capillary vessel spline structure in gel.The condition of aortic annulus modeling more approaches internal milieu, and it has comprised non-endotheliocyte around, as smooth muscle cell and pericyte, also has matrix support.In addition, endotheliocyte is not the cell going down to posterity, and also not in the multiplicative stage, this more can represent the real conditions of the interior angiogenesis of body of current discovery.Therefore, aortic annulus model is comparatively desirable model of angiogenesis, is most widely used detection method in current angiogenesis research.

When research the Molecular Biology Mechanism, sometimes need cell to carry out separation and evaluation, and observation of cell form, but above-mentioned these models all can not reach unicellular research, also can not realize single celled separation.And utilize these models new life's blood vessel, structure is three-dimensional, be difficult to observe clearly with microscope.

Summary of the invention

The shortcoming existing in order to overcome prior art, the purpose of this utility model is on the basis of aortic annulus model, to invent a kind ofly can clearly observe the individual cells of new vessel and to the unicellular instrument that carries out separate study.

In order to achieve the above object, the technical solution adopted in the utility model is: aorta dimensional culture single cell separator, it is characterized in that: comprise two slide glasss, one is common slide glass, its upper surface is with one deck mouse tail collagen, another is porose slide glass, its lower surface is with one deck organic silicon potting adhesive, by treated of two slide glasss with adhered by double sided plaster together, from the hole of porose slide glass, pour into the liquid matrigel of ice bath, make it filling between two slide glasss.After the one side of porose slide glass is processed with organic silicon potting adhesive, had hydrophobic characteristic, cell culture fluid can be stained with this face, so cell can not be attached to this length of looking unfamiliar; And after common slide glass processes with mouse tail collagen, cell take that it is grown for biological support attaches on it.The thickness that gap thickness between two slide glasss is double faced adhesive tape, can be controlled at 0.2-0.5mm, is a space that cell can be grown.

The utility model is the improvement to in-vitro culture model aortic annulus tissue culture, the growth of cell is limited in the space in the middle of two slide glasss, thereby has guaranteed the growth of a cell, is convenient to microscopic examination and takes pictures.

Compared with prior art, advantage is the utility model: this model is the improvement on aortic annulus model basis, has the advantage of aortic annulus model, as real simulation the situation of angiogenesis, operate relatively simply, easily observe; Can also carry out single celled research and separation and Culture, and unicellular being grown on slide glass looks more clear, easily observation.The utility model is mainly used in cultivating the individual cells of new vessel and to the unicellular separate study of carrying out.

Accompanying drawing explanation

Below in conjunction with accompanying drawing, the utility model is described further.

Fig. 1 is STRUCTURE DECOMPOSITION schematic diagram of the present utility model.

Fig. 2 is structural representation of the present utility model.

embodiment

As shown in Figure 1, the utility model comprises two slide glasss that are bonded together with double faced adhesive tape 4, below one be common slide glass 1, its upper surface is with one deck mouse tail collagen, above one be porose 3 slide glass 2, its lower surface, with one deck DOW CORNING organic silicon potting adhesive, is filled with the liquid matrigel of ice bath between two slide glasss.

During making, porose slide glass 2 and common slide glass 1 need dry through cleaning, acid treatment, after alcohol-pickled, at Bechtop middle-ultraviolet lamp, irradiate 2 hours, then in Bechtop, the upper surface of the lower surface of porose slide glass 2 and common slide glass 1 is processed with DOW CORNING organic silicon potting adhesive and mouse tail collagen respectively, by porose slide glass 2 with common slide glass 1 is treated is bonded together with double faced adhesive tape 4 to opposite, use uviolizing 2 hours, now double faced adhesive tape also irradiates together again.Then device bonding, that irradiated is put in culture dish, directly carries out subsequent experimental or sealed membrane sealing, be positioned over 4 degree Refrigerator stores.Carry the day before yesterday and take out for the aortal matrigel of dimensional culture from-20 degree refrigerators, the hole 3 by it from porose slide glass 2 injects for the aortal matrigel of dimensional culture, by syphonic effect, makes it filling between two slide glasss.

During use, in Bechtop, the aorta of rat is taken out, PBS cleans, and cuts into pieces, is put in the hole 3 of porose slide glass 2, this is installed together with culture dish at 37 degrees Celsius, in the incubator of 5%CO2, hatch 20 minutes, after matrigel solidifies, withdrawing device and culture dish, in Bechtop, add nutrient solution, put back to again in incubator, continue to cultivate 3 to 7 days, every one day, change a not good liquor.From the aorta cell that dissociates, can between two slide glasss, grow, within about about one week, can form new vessel.What porose slide glass 2 was coated with is hydrophobic organic joint sealant, and cell can be in this length of looking unfamiliar, and can only be grown on the common slide glass 1 of processing with mouse tail collagen.Directly visual inspection or with the cell of growing on common slide glass in microscopic examination culture dish and take pictures, also this utility model can be taken out from culture dish, porose slide glass 2 is removed, carry out acridine orange dyeing, immunocytochemical stain and in Bechtop, carry out the subsequent operationss such as separation and Culture.The thickness of double faced adhesive tape 3, the distance between two slide glasss is controlled between 0.2-0.5mm, only allows the growth of individual cells, thereby guarantees that the cell of studying is unicellular, the cell that last separation and Culture obtains is mono-clonal, and individual cells is convenient to clearly observe and take pictures.

Claims

1. aorta dimensional culture single cell separator, it is characterized in that: comprise two slide glasss that are bonded together with double faced adhesive tape (4), below one be common slide glass (1), its upper surface is with one deck mouse tail collagen, above one be the slide glass (2) of porose (3), its lower surface, with one deck DOW CORNING organic silicon potting adhesive, is filled with the liquid matrigel of ice bath between two slide glasss.

2. aorta dimensional culture single cell separator according to claim 1, is characterized in that, the spacing of two slide glasss is 0.2-0.5mm.