CN2760041Y - Miniaturized cultivation unit for zooblast - Google Patents
Miniaturized cultivation unit for zooblast Download PDFInfo
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- CN2760041Y CN2760041Y CN 200420081459 CN200420081459U CN2760041Y CN 2760041 Y CN2760041 Y CN 2760041Y CN 200420081459 CN200420081459 CN 200420081459 CN 200420081459 U CN200420081459 U CN 200420081459U CN 2760041 Y CN2760041 Y CN 2760041Y
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Abstract
The utility model discloses a miniaturized cultivating device for animal cells, which is composed of a silicone tube which is made of a ventilation silica gel, a ventilation silica gel plug and a hollow fiber silk. The cells are covered in the hollow fiber silk with collagen gel, and because the miniaturized cultivating device of the animal cells has the abilities to transfer oxygen and mass, which are suitable for the survival of the cells, the functions of the drug metabolism of the cells, etc., are maintained. By combining the microminiaturization advantage of a pore plate and the three-dimensional bionic culture advantage of an artificial liver reactor, the bioactivity of the animal cells are maintained. The utility model is used as a model out of the liver cell of the animal which is researched through the drug metabolism and pharmacological toxicology and is used for laboratory research on the generation of cell culture condition and production. The utility model can be used for a carrier which is researched through the animal cell culture condition, and lays the foundation of industrialized production.
Description
Technical field
The utility model relates to a kind of microminiaturized incubator of zooblast.
Background technology
The research of existing medicine pharmacological toxicology is based on three kinds on animal model, external primary cell model and continuous cell line model.Animal model has experimental period long, and efficient is low, and individual difference causes result error excessive greatly, and be unfavorable for shortcomings such as protection of animal, and can not eliminate species variation, be the fatal weakness of animal model.Though the continuous cell line training method is simple, efficient is expensive low, and continuous cell line and primary cultured cell still have on 26S Proteasome Structure and Function than big difference, can not embody medicine fully at human body or intravital metabolism of animal and toxic action.
At present, the investigator begins to pay attention to the application of external primary cell model on drug research both at home and abroad.Its required cell concentration is few, may fundamentally eliminate species variation and individual difference, and interfering factors is little, the good reproducibility as a result of gained.The cell in vitro model that is used for drug research mainly contains orifice plate cultivation and plate cultivation etc., though the mode of this kind two dimension is simple to operate, keep the cytoactive time short, cell function is easily lost, the good 26S Proteasome Structure and Function of analogue body inner cell, these defectives are restricted its application.And the dimensional culture mode, as tubular fibre bioartificial liver reactor, though can the long period keep cell function, the simulated liver environment, but complex structure needs the circulation of multichannel oxygen supply and substratum, and is bulky, cost is also quite high, obviously is not suitable for the drug metabolism study and the high-throughout drug screening that need miniaturization.
Simultaneously, laboratory study cell culture condition and production products of cellular metabolism need a kind of miniaturization, the device that is fit to carry out condition experiment, optimization, screening.And the large-scale hollow fiber reactor that industrial production is used obviously can't meet this requirement.If can find a kind of both simple to operately, the training method of amplifying easily again has certain meaning for the early-stage Study of zooblast suitability for industrialized production.
Summary of the invention
In order to overcome the deficiency of existing drug research model, gain enlightenment from bioartificial liver's reactor and artificial organs, the purpose of this utility model is to provide a kind of microminiaturized incubator of zooblast, required structure of general reactor and operation have been simplified greatly, mode with dimensional culture reaches the bionics effect, and can long term maintenance zooblast function.
The technical scheme that its technical problem that solves the utility model adopts is: comprise silicone tube, silica gel plug, the hollow fibre filament more than 1; Hollow fibre filament is contained in the silicone tube, and seal with silica gel plug at silicone tube two.
Said silicone tube and silica gel plug material are for passing the oxygen silica gel material.
The material of said hollow fibre filament is polypropylene, polysulfones, polyacrylonitrile or the polyethylene of its molecular weight cut-off at 100KDa～1000KDa; The hollow fibre filament hollow fibre filament is the 1-100 root.
The volume of said microminiaturized incubator is 0.1ml-10ml.
The useful effect that the utlity model has is: can carry out long-term dimensional culture to zooblast, the biological activity that has kept zooblast, the one, as the animal liver cell external model of the research of drug metabolism and pharmacological toxicology, the 2nd, be used for the laboratory study that cell culture condition and product are produced.And can be used as the carrier of animal cell culture condition research, for suitability for industrialized production lays the foundation.
Description of drawings
Fig. 1 is a structural representation of the present utility model;
Fig. 2 is albumin and the urea production curve of embodiment 3.
Embodiment
The utility model is described in further detail below in conjunction with drawings and Examples.
As shown in Figure 1, the utility model comprises: silicone tube 1, the silica gel plug hollow fibre filament 3 more than 2,1; Hollow fibre filament 3 is contained in the silicone tube 1, and seal with silica gel plug 2 at silicone tube 1 two.
The comparison that microminiaturized incubator of specific embodiment 1:(and plate are cultivated)
Silicone tube 1 long 13cm, internal diameter 0.8cm, external diameter 1.2cm.Silica gel plug 2 thin end 0.75cm, butt end 1.35cm.Hollow fibre filament 3 polyacrylonitrile materials, external diameter 0.110cm-0.120cm, molecular weight cut-off are 1000KDa.
The results motility rate is higher than 90% rat hepatocytes by 10
6Cells/ml density was inoculated in 4 times of enrichment mediums and mouse tail collagen solution mixed solution (1: 3; V: v), inject 3,37 ℃ of placements of hollow fibre filament and made it to solidify in 10 minutes.Hollow fibre filament 3 is cut short, 8cm * 12 piece, the silicone tube 1 of packing into adds 5ml and contains 5% foetal calf serum Williams ' E substratum, puts into 37 ℃, 5%CO
2Incubator leaves standstill cultivation.
Will be with batch rat hepatocytes by 0.8 * 10
6Cells/ml inserts 5ml and contains 5% foetal calf serum Williams ' E substratum, cultivates at the plate that is covered with mouse tail collagen.Culture condition is same as described above.
Treat in the plate behind the cell attachment (12-18h), in microminiaturized incubator and plate, add respectively and contain medicine 10mM acetaminophen substratum to be measured and do not contain medicine substratum (as blank).Thereafter the substratum that every 60h more renews.
Each repeats three groups, measures liver function index urea and albumin, the observation of cell form.
The result is as shown in table 1 below.
Table 1:
| Time | Dosing 60h | Dosing 120h | ||
| Group | Microminiaturized incubator | Plate | Microminiaturized incubator | Plate |
| Urea production is than (blank group/acetaminophen group) | 1.76 | 1.30 | 1.69 | 1.24 |
| Albumin rate ratio (blank group/acetaminophen group) | 1.30 | 1.11 | 3.67 | 1.23 |
As seen from the above table, the existing state of rat hepatocytes in microminiaturized incubator is better than plate monolayer culture, and microminiaturized incubator group is more remarkable than plate group to the reaction of liver toxicity medicine acetaminophen, has illustrated that microminiaturized incubator more is applicable to drug toxicity research than plate.
General porous plate often is used as the carrier of cells in vitro, and porous plate is cultivated and the plate cultivation all is the monolayer culture mode of two dimension, and a lot of common ground is arranged.Therefore, experiment finds that microminiaturized incubator culturing cell is more superior than plate, and the difference of the utility model and porous plate also can be described.
The microminiaturized incubator of specific embodiment 2:(is used for the research of drug metabolism)
Silicone tube 1 long 13cm, internal diameter 0.8cm, 1.2cm.Silica gel plug 2 thin end 0.75cm, butt end 1.35cm.Hollow fibre filament 3 polysulfones materials, external diameter 0.110cm-0.120cm, molecular weight cut-off are 1000KDa.
The results motility rate is higher than 90% rat hepatocytes by 10
6Cells/ml density was inoculated in 4 times of enrichment mediums and mouse tail collagen solution mixed solution (1: 3; V: v), inject 3,37 ℃ of placements of hollow fibre filament and made it to solidify in 10 minutes.Hollow fibre filament 3 is cut short, 8cm * 12 piece, the silicone tube 1 of packing into adds 5ml and contains 5% foetal calf serum Williams ' E substratum, puts into 37 ℃, 5%CO
2Incubator leaves standstill cultivation.
Will be with batch rat hepatocytes by 0.8 * 10
6Cells/ml inserts 5ml and contains 5% foetal calf serum Williams ' E substratum, cultivates at the plate that is covered with mouse tail collagen.Culture condition is same as described above.
After cultivating 60h, each group adds the Phenacetin solution of about 40uM, and through the drug metabolism of 3h, the HPLC method is measured the content of Phenacetin and meta-bolites acetaminophen.The result is as shown in table 2:
Table 2:
| Time | Sampling Oh | Sampling 3h | ||
| Group | Microminiaturized incubator | Plate | Microminiaturized incubator | Plate |
| Phenacetin (g/L) | 0.081 | 0.081 | 0.035 | 0.066 |
| Acetaminophen (g/L) | 0 | 0 | 0.0097 | 0.0048 |
As seen from the above table, rat hepatocytes is higher than the plate culturing cell in the ability of microminiaturized incubator intracellular metabolite Phenacetin, illustrates that Cytochrome P450 specific activity plate monolayer culture is good, and therefore microminiaturized incubator more is applicable to drug metabolism study than plate.
The microminiaturized incubator of specific embodiment 3:(is used for the research of use in medicament-induced hepatotoxicity)
Silicone tube 1 long 13cm, internal diameter 0.8cm, external diameter 1.2cm.Silica gel plug 2 thin end 0.75cm, butt end 1.35cm.Hollow fibre filament 3 polysulfones materials, external diameter 0.110cm-0.120cm, molecular weight cut-off are 1000KDa.
The results motility rate is higher than 90% rat hepatocytes was inoculated in 4 times of enrichment mediums and mouse tail collagen solution mixed solution (1: 3 by 106cells/ml density; V: v), inject 3,37 ℃ of placements of hollow fibre filament and made it to solidify in 10 minutes.Hollow fibre filament 3 is cut short, 8cm * 12 piece, the silicone tube 1 of packing into adds 5ml and contains 5% foetal calf serum Williams ' E substratum, puts into 37 ℃, 5%CO
2Incubator leaves standstill cultivation.
After cultivating 12-18h, each group changes to respectively that to contain drug level be Rifampin 10mg/ml, the substratum of vazadrine 15mg/ml and do not contain the substratum of medicine, and every 24h takes a sample once, and every 60h changes substratum once.Triplicate, the urea of working sample and albumin output.The result as shown in Figure 2, the albumin growing amount is change curve (left side) in time, urea production is change curve (right side) in time.
As seen from the figure, the effect of the Rifampin of this concentration and vazadrine down, the output of albumin and urea all is lower than the blank group, so medicine has damaging action to rat hepatocytes, thereby causes the part forfeiture of cell liver function.Illustrated that the utility model is used for the feasibility of liver toxicity drug research.
The microminiaturized incubator of specific embodiment 4:(is used for the cultivation of successive cell strain)
Silicone tube 1 long 13cm, internal diameter 0.8cm, 1.2cm.Silica gel plug 2 thin end 0.75cm, butt end 1.35cm.Hollow fibre filament 3 polypropylene materials, external diameter 0.110cm-0.120cm, molecular weight cut-off are respectively 30KD and 100KD.
L cell L929 is pressed 10
6Cells/ml density was inoculated in 4 times of enrichment mediums and mouse tail collagen solution mixed solution (1: 3; V: v), inject 3,37 ℃ of placements of aforesaid two kinds of hollow fibre filaments and made it to solidify in 10 minutes.Hollow fibre filament 3 is cut short, 8cm * 12 piece, the silica gel pipe shaft 1 of packing into adds 5ml and contains 10% calf serum, 1640 substratum, puts into 37 ℃, 5%CO
2Incubator leaves standstill cultivation.
The growing state (cell proliferation quantity) of L929 cell in the microminiaturized incubator of molecular weight cut-off 100KD is better than the molecular weight cut-off 30KD of identical material.Thereby screening obtains 100KD polysulfone hollow fibre silk and is suitable for cell cultures.
The microminiaturized incubator of specific embodiment 5:(is used for the screening of hepatic)
Silicone tube 1 long 13cm, internal diameter 0.8cm, external diameter 1.2cm.Silica gel plug 2 thin end 0.75cm, butt end 1.35cm.Hollow fibre filament 3 polysulfones materials, external diameter 0.110cm-0.120cm, molecular weight cut-off are 1000KDa.
The results motility rate is higher than 90% rat hepatocytes by 10
6Cells/ml density was inoculated in 4 times of enrichment mediums and mouse tail collagen solution mixed solution (1: 3; V: v), inject 3,37 ℃ of placements of hollow fibre filament and made it to solidify in 10 minutes.Hollow fibre filament 3 is cut short, 8cm * 12 piece, the silicone tube 1 of packing into adds 5ml and contains 5% foetal calf serum Williams ' E substratum, puts into 37 ℃, 5%CO
2Incubator leaves standstill cultivation.
After cultivating 12-18h, each group changes to respectively that to contain drug level be 10mM acetaminophen substratum, 10mM acetaminophen+4mM gsh, 10mM acetaminophen+4mM N-acetylcystein, 10mM acetaminophen+4mM halfcystine and do not contain medicine substratum (as blank).
Observation of cell form behind the 24h finds that the cell motility rate of 10mM acetaminophen+4mM gsh group is higher.Promptly from three kinds of hepatics to be selected, screen the liver lesion induced by drugs that obtains the gsh Abensanil provide protection is arranged.This is the example that the utility model is used for the hepatic screening.
Claims (4)
1. the microminiaturized incubator of a zooblast is characterized in that: comprise silicone tube (1), silica gel plug (2), the hollow fibre filament more than 1 (3); Hollow fibre filament (3) is contained in the silicone tube (1), and seal with silica gel plug (2) at silicone tube (1) two.
2. the microminiaturized incubator of a kind of zooblast according to claim 1 is characterized in that: said silicone tube (1) and silica gel plug (2) material are for passing the oxygen silica gel material.
3. the microminiaturized incubator of a kind of zooblast according to claim 1, it is characterized in that: the material of said hollow fibre filament is polypropylene, polysulfones, polyacrylonitrile or the polyethylene of its molecular weight cut-off at 100KDa-1000KDa; Hollow fibre filament (3) is the 1-100 root.
4. the microminiaturized incubator of a kind of zooblast according to claim 1, it is characterized in that: the volume of said microminiaturized incubator is 0.1ml-10ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200420081459 CN2760041Y (en) | 2004-08-01 | 2004-08-01 | Miniaturized cultivation unit for zooblast |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200420081459 CN2760041Y (en) | 2004-08-01 | 2004-08-01 | Miniaturized cultivation unit for zooblast |
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| CN2760041Y true CN2760041Y (en) | 2006-02-22 |
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| CN 200420081459 Expired - Fee Related CN2760041Y (en) | 2004-08-01 | 2004-08-01 | Miniaturized cultivation unit for zooblast |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105733944A (en) * | 2016-05-04 | 2016-07-06 | 浙江大学 | Construction method for six-pore plate hepatic cell hollow fiber reactor |
-
2004
- 2004-08-01 CN CN 200420081459 patent/CN2760041Y/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105733944A (en) * | 2016-05-04 | 2016-07-06 | 浙江大学 | Construction method for six-pore plate hepatic cell hollow fiber reactor |
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|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |