CN1249217C - Cell culture incubator - Google Patents
Cell culture incubator Download PDFInfo
- Publication number
- CN1249217C CN1249217C CN 03102404 CN03102404A CN1249217C CN 1249217 C CN1249217 C CN 1249217C CN 03102404 CN03102404 CN 03102404 CN 03102404 A CN03102404 A CN 03102404A CN 1249217 C CN1249217 C CN 1249217C
- Authority
- CN
- China
- Prior art keywords
- culture
- cell
- culture bag
- substratum
- bottle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Abstract
The present invention relates to an improved cell culture device, particularly to an in-vitro culture device for mammal cells and a method thereof. The present invention is characterized in that the in-vitro culture device for mammal cells is mainly composed of a culture bottle shell body and a separated cell culture bag, wherein the separated cell culture bag is arranged in the culture bottle shell body and is composed of a selective permeation membrane, and the culture bag limited by the selective permeation membrane is completely immersed in a liquid culture medium in a culture bottle. The upper wall of the culture bottle is provided with a plurality of ventilating holes.
Description
Invention field
The cell culture apparatus that the present invention relates to improve particularly relates to, but is not limited to, and the vitro culture various types of cells comprises mammalian cell, insect cell, the culture apparatus of vegetable cell etc. and tissue culture and method.Be characterised in that said device mainly places the cell culture bags of the separation that is made of permselective membrane of its inside to constitute by culturing bottle housing and one, wherein be immersed in fully in the liquid nutrient medium in the culturing bottle, and the upper wall of culturing bottle has a plurality of ventilating pits by the culture bag of permselective membrane restriction.Culturing bottle of the present invention can use separately, also can constitute equality circuit on shaking table or rotated bed by a plurality of culturing bottles, to realize the production in enormous quantities of cell and its useful products.
Background of invention
The device of cultured cell in vitro and tissue is class biological experiment and a production equipment commonly used.Traditional cell in vitro particularly mammalian cell is cultivated and normally to be carried out in fixed tissue culture flasks or rolling bottle.When using this culture apparatus, regularly change substratum, remove refuse and collect useful product.There are shortcomings such as troublesome poeration, cell density is little, condition (as nutrient concentration) is wayward, the cell or tissue production concentration is lower and be easy to lose in these culture apparatuses.
United States Patent (USP) 4,748 describes a kind ofly can see through gas but cell cultures compartment that liquid-tight lamella and top dialysis membrane limit by the bottom for No. 124.Though this structure can make dialysis membrane not get clogged, exist to change oxygen pressure limited in one's ability, the surface chemistry of gas permeable material is required high and is difficult to realize shortcoming such as high-density culture.United States Patent (USP) 5,693, the Tissue Culture Flask that discloses a kind of separation No. 537, being characterised in that has a cell that is made of semi-permeable membranes to isolate culturing room in the bottle, and the air-permeable envelope below the culturing room forms culturing bottle bottom, directly ingress of air; The lamella of top selective permeation compound then contacts with nutrient solution.An air inlet and charging opening are arranged at culturing bottle top, and air enters culturing room by the bottle bottom, form circulation with inlet mouth again, to allow carrying out gaseous interchange by bottom air-permeable envelope and the content of separating culturing room.This device makes the nutrient solution relative separation outside cell expression product and the culturing room, and substratum or gas can enter culturing room by semi-permeable membranes, changes purpose thereby reach nutrition.Yet this United States Patent (USP) still exists the cell culture chamber of separation to have only that one side contacts nutritional medium, the bottom ventilating pit is easily blocked and well such as is easy to be polluted at shortcoming.
In order to address these problems, be necessary further to improve on the basis of existing technology the structure of culture apparatus, in the hope of nutrient substance supply, increasing gas exchange capacity, the raising cultured cells density that improves unit volume, and reduce opportunities for contamination as much as possible, thereby reach the purpose of raising the efficiency, improving product quality and reduce production costs.
Goal of the invention
Therefore, one object of the present invention provides a kind of cell culture apparatus of separation, said culture apparatus has the culturing bottle of ventilating pit by its upper wall and places the cell culture bags of one of bottle inside separation that is made of permselective membrane and the network that is used to support said culture bag, wherein is immersed in fully in the liquid nutrient medium in the culturing bottle by the culture bag of permselective membrane restriction.
According to a preferred embodiment of the invention, the said permselective membrane that wherein constitutes culture bag is can see through gas and can see through semi-permeable membranes less than the compound of the specific molecular weight range of selecting.
According to a preferred embodiment of the invention, wherein the culture bag support be arranged at culture bag below, contact with the bottom surface of said culture bag with dissolved gases wherein to allow substratum, and the culture bag of freely coming in and going out.
According to a preferred embodiment of the invention, charge into wherein that the height of substratum liquid level is not less than 15 millimeters behind the substratum in the culturing bottle.
According to a particularly preferred embodiment of the present invention, wherein the end face left and right sides of said culturing bottle has at least 1 ventilating pit respectively.
According to a preferred embodiment of the invention, wherein an end of culturing bottle has one directly to communicate with the culture bag of culturing bottle inside and to first inlet of wherein importing cell or tissue piece suspension, and the other end have one lead to bottle inner to and wherein import second of substratum and enter the mouth.
According to a particularly preferred embodiment of the present invention, the outside port of wherein said first inlet is funnelform.
According to a preferred embodiment of the invention, wherein an end opening of culture bag directly links to each other with the inboard port down of first entrance extension to culturing bottle bottle inside, and the other end is that blind end or available dialysis tubing clamp the opening that closes.
Another object of the present invention provides a kind of method of culturing cell, and this method comprises by the penetrating film that can see through gas and optionally see through less than the compound of specific molecular weight range and constitute culture bag, places it on the network that is positioned at below it; With a small amount of substratum that culture bag is wetting, funnel-form first inlet by culturing bottle injects cell or tissue piece suspension then, residual air rear enclosed first inlet in the venting bag, and inject substratum to specific horizontal plane by second inlet is so that substratum can the whole culture bag of submergence; Under predetermined temperature, the cell of inoculation is kept the specific time in culture bag, allow simultaneously environment in the culture bag with its on every side substratum and carry out exchanging of nutrient substance, products of cellular metabolism and gas by substratum with atmospheric environment.
Brief Description Of Drawings
Fig. 1 is the front view of separation cell culture apparatus of the present invention.
Fig. 2 is the side-view that is prolonging major axis medullary ray longitudinal section of cell culture apparatus of the present invention.
More than all have unified Ref. No. (seeing for details hereinafter) in each accompanying drawing.
The detailed description of invention
The cell culture apparatus that the present invention relates to improve particularly relates to, but is not limited to, and in vitro culture is all kinds of thin Born of the same parents comprise mammalian cell, insect cell, culture apparatus and side that plant cell etc. and tissue are cultivated Method. Be characterised in that said blake bottle housing is built-in with the cell training of a separation that is made of permselective membrane Support bag and be used for supporting the support of said culture bag, and soaked fully by the culture bag of permselective membrane restriction Not in the fluid nutrient medium in blake bottle. Improvements over the prior art of the present invention and its advantage comprise: will be equipped with The selective culture bag of cell or tissue is tiled on the specific support, to guarantee outside bag inner cell or tissue and the bag Environment carries out the exchange of sufficient nutrition and gas; The far-end of said cell culture bags can be closed, also Can be open, and when using can by dialysis sackholder or similar device sealing said distal openings. Training Support the bottle top and be added with a plurality of gaseous exchanges hole, get clogged to prevent pore; In device, inculcate the suitable for reading of cell and be Funnel-form, thus avoided retaining or the anti-excessive opportunities for contamination that causes because of liquid.
An object of the present invention is to provide a kind of for cultured cell particularly mammalian cell or tissue The culture apparatus of separating. According to a preferred embodiment of the invention, the cell or tissue of said separation training Foster device consists essentially of by seeing through gas and less than the penetrating film restriction of the compound of specific molecular cut off And the culture bag of holding the cell or tissue suspension, and be enclosed in said culture bag outside and be filled with liquid The platypelloid type blake bottle of culture medium consists of. Wherein an end of blake bottle has one directly and the cultivation of blake bottle inside Bag communicates and to first entrance of wherein importing cell or tissue piece suspension, and the other end has one to lead in the bottle Section is to second entrance of also wherein importing culture medium. The outboard end of said first entrance is funnelform. Cultivate This funnel-shaped structure of bottle bottle top entrance can prevent and reduce because of liquid retaining or instead overflow effectively The pollution that may cause. Particularly, the top of the blake bottle of culture apparatus of the present invention part has more than 2 and can protect Culture medium carries out the passage that gas exchanges with external environment in the card bottle. Like this, in blake bottle, be filled with culture medium After, can see through penetrating film from being full of by the cell or tissue culture in the culture bag of permselective membrane restriction Obtain the various nutritional labelings that required comprising is dissolved in oxygen wherein in the culture medium of surrounding environment, side by side Go out useful metabolite or refuse.
Permselective membrane (ventilated membrane) material that is used for the formation cell culture bags can be to be Growth of Cells and numerous Grow hydrophilic or hydrophobic, porous or the non-porous material of any biocompatibility of the pericellular environment that provides suitable Material. Can be cellulose, poly-carbonic acid, polyacrylamide etc. such as said material. Assorted when being used for cultivating mouse When handing over oncocyte, dialysis membrane answers molecular cut off to be not less than 15,000 daltonian molecule and compounds usually. In addition, be worth should be mentioned that the two ends of cell culture apparatus of the present invention respectively are provided with an entrance and establish up A plurality of passages are arranged, thus not only can allow suction pipe to enter blake bottle or culture bag inside, and can prevent because Temperature increase and in the bottle that causes or the bag internal pressure increase. Particularly, the structure of apparatus of the present invention has prevented because steaming Send out dislysate caused and lose, and can keep for a long time the high-density growth of cell.
The present invention is the most important improvement of prior art, cell in the prior art is cultivated compartment only have one The nutrition exchange is carried out by pellicle and culture medium in individual side, all can in all sides that change cell cultivation compartment into Carry out the nutrition exchange by pellicle and culture medium, thereby greatly improved nutrition supply and the growth and breeding of cell Condition.
What should particularly point out is, although cell or tissue culture apparatus of the present invention is not provided with an independently gas Body is filled with part, but the inventor believes, owing to have equably 2 on the upper side wall of blake bottle in this device Individual above passage can realize fully that by these passages ambient atmosphere environment or people are the hyperoxia that causes The gas of culture medium exchange around environment and the culture bag, and rely on the oxygen that is dissolved in the culture medium to guarantee in the bag The oxygen supply of cell or tissue. Hybridoma and lotus that our laboratory uses device of the present invention to finish have matter The culture experiment such as CHO of grain advantageously prove applicability (the antibody producing amount of putting into practice of apparatus of the present invention Can reach 1-5mg/ml).
Another object of the present invention provides a kind of method of cultured cell, and the method comprises by seeing through gas also Optionally see through the culture bag of penetrating film formation less than the compound of specific molecular weight range, with the one end On the network of opening below the inner port of first entrance tightly engages and be held in place it; Use a small amount of training It is wetting with culture bag to support base, and funnel-form first entrance by blake bottle injects cell or tissue piece suspension, row then Go to close first entrance behind the residual air in the bag, and inject culture medium to specific horizontal plane by second entrance, So that culture medium can the whole culture bag of submergence; Under predetermined temperature, the cell of inoculation is protected in culture bag Hold the specific time, allow simultaneously environment in the culture bag and culture medium around it and by culture medium and big compression ring Exchanging of nutrient, products of cellular metabolism and gas carried out in the border.
Also it should be noted that under some particular case, in order to carry out the cultivation in enormous quantities of cell, can be with many Individual blake bottle of the present invention be fixed on simultaneously can according to the forward or backwards rotation of speed of selecting (for example 1-5 second/ Turn to) or the single or multiple lift fixed mount or bed body of oscillation in the pitch mode (for example 1-5 time/second) on, to realize cell Continuous Cultivation in enormous quantities and the production of cell expression product.
Embodiment
In order to further describe the present invention,, the structure and the working order of apparatus of the present invention is described more specifically below with reference to accompanying drawing as embodiment.
Fig. 1 is the front view of separation cell culture apparatus of the present invention.Wherein 1 is the culturing bottle bottle, and its shape, size and making material are all similar to traditional common culturing bottle.The 2nd, culturing bottle bottle one distal process goes out directly communicates with the culture bag 4 of culturing bottle inside and to first entering the mouth of wherein importing cell or tissue piece suspension (substratum that promptly contains cell or tissue) 6.Culture bag is the cell cultures place, wherein deposits the substratum 6 that contains cell or tissue, and covers to first inlet, 2 tegmentums 201 of wherein introducing cell or tissue.Wherein 3 be the culturing bottle the other end be that J-shaped stretches out lead to bottle inner to and wherein import second inlet of substratum 7.Second inlet, 3 tegmentums 301 cover.The 5th, the ventilating pit that culturing bottle housing upper wall is offered under the horizontal state, generally each device has 2-8 the ventilating pit that the aperture is generally 5-20mm.For fear of contingent pollution, can the upper limb of ventilating pit 5 additional one airtight, intransitable air filter membrane of most of microbe or filter disc.
Fig. 2 is the side-view that is prolonging major axis medullary ray longitudinal section of cell culture apparatus of the present invention.Can more be well understood to the internal structure of device from Fig. 2: the part that the upper limb of first inlet 2 accounts for this inlet top 1/4 is big mouthful of funnel shaped that makes progress, and such structure can help avoiding or reduce in application of sample or the culturing process because of liquid hold-up or the anti-pollution of overflowing and being caused.The parts that first inlet 2 stretches to the bottom about 1/2 in the culturing bottle are for the upside down funnel shape that Open Side Down or straight shape, and its lower edge bore is equivalent to the bore of first inlet 2 approximately.Unique opening of culture bag 4 and first inlet, 2 lower edges that stretch to the upside down funnel shape part 202 of culturing bottle inside connect airtight.The other end is the good to eat of dialysis sackholder 401 clampings closure or available.The below of culture bag 4 is the network 8 that places the about 2-4mm in aperture on the culturing bottle lower wall, is used to support the culture bag that is made of permselective membrane, and guarantees that substratum 7 can contact with the culture bag bottom surface in the culturing bottle.
In the operation, the substratum 6 that contains cell or tissue is introduced in the culture bag 4 by first inlet 2.Simultaneously, liquid nutrient medium 7 is introduced in the bottle of culturing bottle 1, till the whole culture bag 4 of substratum 7 submergences in culturing bottle 1 by second inlet 3.In general, after charging into liquid nutrient medium in culturing bottle and the culture bag respectively or containing the substratum of cell or tissue, the substratum headspace of substratum top is not less than 1/3 of smart total height in the culturing bottle in the culturing bottle, thereby guarantees to have in this space q.s by ventilating pit 5 and substratum 6 and 7 and and then the gas that exchanges by these substratum and cell.
Kai Fang ventilating pit 5 is as the communication channel of environment in the culturing bottle and ambient atmosphere environment in fact, it not only helps importing gas in culturing bottle, guarantee the oxygen supply and the gaseous interchange of cell, and can prevent the interior environmental stress increase that Yin Wendu raises and caused in the cell cultivation process effectively.In addition, the existence of ventilating pit and liquid nutrient medium superjacent air space also can be avoided the reduction of liquid nutrient medium oxygen carrying capacity, and can avoid because of pressure increases the extruding that culture bag is caused, to guarantee the hydrostaticpressure balance between culture bag and culturing bottle.
Permselective membrane (dialysis membrane) material that is used to constitute cell culture bags can be hydrophilic or hydrophobic, porous or the non-porous material that any biocompatibility of suitable pericellular environment is provided for cell growth and breeding.For example can be Mierocrystalline cellulose, poly-carbonic acid, polyacrylamide etc.When being used to cultivate mouse hybridoma cell, the general molecular weight that keeps is greater than 15,000 daltonian molecule or compounds in the dialysis membrane.Film with this feature can make the antibody of cell, the somatomedin that adds and emiocytosis all be retained in the culture bag, and the products of cellular metabolism that has than small molecular weight then is rejected to outside the culture bag.Under some specific situation, those skilled in the art can select to have the film of PSPP scope fully according to actual needs.
Be filled with in culturing bottle 1 under the situation of liquid nutrient medium, the substratum 6 introducing cell culture bags 4 that will contain cell or tissue need enough pressure and overcome the hydrostaticpressure that the outer substratum of bag forms.For this reason, lead to culture bag first the inlet should be enough in culture bag, to stretch into a suction pipe, syringe or other medium containers.In addition,, the cell or tissue suspension in the culture bag contacts, so the problem that this device does not have substratum evaporation or dialysis to be restricted yet with substratum 7 because always seeing through semi-permeable membranes.
The penetrating mould material that constitutes culture bag 4 is the biocompatible materials that can see through gas and specified range compound molecular weight, for example can be maybe can not see through liquid, hydrophilic or hydrophobic, porous or non-porous, the high polymer material of the about 0.1-0.5mm of thickness.When selecting penetrating mould material, generally the principal element that should consider is the interaction between the protein molecule of the gas permeable of film and film and cell or ad hoc structure.
The material of making culturing bottle 1 can be any biocompatible materials, and preferably has certain transparency, and so promptly the pH of possible naked-eye observation substratum changes and contingent microbial contamination.Preferred culturing bottle material is a polystyrene.
Claims (6)
1, a kind of cell culture apparatus of separation, said culture apparatus has the culturing bottle of ventilating pit by its upper wall and the network that places the cell culture bags of one of bottle inside separation that is made of permselective membrane and be used to support said culture bag constitutes, wherein be immersed in fully in the liquid nutrient medium in the culturing bottle by the culture bag of permselective membrane restriction, the liquid level that is characterised in that substratum in (1) culturing bottle is not less than 15 millimeters, permselective membrane is immersed in fully cultivates in the flat interior liquid nutrient medium; (2) end of culturing bottle has one directly to communicate with the culture bag of culturing bottle inside and be funnelform first inlet to the outside port of wherein importing cell or tissue piece suspension, and the other end have one lead to bottle inner to and wherein import second of substratum and enter the mouth; And (3) have at least 1 ventilating pit respectively in the culturing bottle end face left and right sides.
2, according to the cell culture apparatus of claim 1, the said permselective membrane that wherein constitutes culture bag is can see through gas and can see through semi-permeable membranes less than the compound of the specific molecular weight range of selecting.
3, according to the cell culture apparatus of claim 1, wherein the culture bag support be arranged at culture bag below, contact with the bottom surface of said culture bag with dissolved gases wherein to allow substratum, and the culture bag of freely coming in and going out.
4, according to the cell culture apparatus of claim 1, wherein an end opening of culture bag directly links to each other with the inboard port down of first entrance extension to culturing bottle bottle inside, and the other end is that blind end or available dialysis tubing clamp the open end of closing.
5, a kind of method of culturing cell, this method comprise by the penetrating film that can see through gas and optionally see through less than the compound of specific molecular weight range and constitute culture bag, places it on the network that is positioned at below it; With a small amount of substratum that culture bag is wetting, funnel-form first inlet by culturing bottle injects cell or tissue piece suspension then, residual air rear enclosed first inlet in the venting bag, and inject substratum to specific horizontal plane by second inlet is so that substratum can the whole culture bag of submergence; Under predetermined temperature, the cell of inoculation is kept the specific time in culture bag, allow simultaneously environment in the culture bag with its on every side substratum and carry out exchanging of nutrient substance, products of cellular metabolism and gas by substratum with atmospheric environment.
6, method according to claim 5, said culturing bottle can use separately, also a plurality of culturing bottles can be fixed on simultaneously can according to the speed of selecting rotate forward or backwards or the single or multiple lift anchor or bed body of oscillation in the pitch mode on, to carry out cultured continuously in enormous quantities.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03102404 CN1249217C (en) | 2003-01-27 | 2003-01-27 | Cell culture incubator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03102404 CN1249217C (en) | 2003-01-27 | 2003-01-27 | Cell culture incubator |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1521253A CN1521253A (en) | 2004-08-18 |
| CN1249217C true CN1249217C (en) | 2006-04-05 |
Family
ID=34281706
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03102404 Expired - Lifetime CN1249217C (en) | 2003-01-27 | 2003-01-27 | Cell culture incubator |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1249217C (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101668843A (en) * | 2007-04-27 | 2010-03-10 | 东洋制罐株式会社 | Cell culture apparatus, cell culture system and cell culture method |
| DE102008049120A1 (en) * | 2008-09-26 | 2010-04-01 | Bayer Technology Services Gmbh | Method for reducing deposits in the cultivation of organisms |
| CN101993823B (en) * | 2009-08-28 | 2012-11-07 | 中国航天员科研训练中心 | Environmental self-control type cytology experimental platform |
| ES2435092B1 (en) * | 2012-06-14 | 2014-09-09 | Aglaris Cell S.L. | Method and cell culture system |
| JPWO2016027800A1 (en) * | 2014-08-22 | 2017-06-29 | オリンパス株式会社 | Cell culture bag, cell culture device and cell culture container |
| US10655097B2 (en) * | 2014-12-22 | 2020-05-19 | Saint-Gobain Performance Plastics Corporation | T-cell culture double bag assembly |
| CN106085850A (en) * | 2016-06-28 | 2016-11-09 | 上海闪锦生物科技有限公司 | A kind of culture bottle improving cell amplification speed |
| CN106967683A (en) * | 2017-05-09 | 2017-07-21 | 林涛 | The method for cultivating pluripotential hemopoietic stem cell |
-
2003
- 2003-01-27 CN CN 03102404 patent/CN1249217C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CN1521253A (en) | 2004-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210253997A1 (en) | Highly efficient gas permeable devices and methods for culturing cells | |
| US11905506B2 (en) | Multilayered cell culture apparatus | |
| AU2011200410B2 (en) | Cell culture methods and devices utilizing gas permeable materials | |
| US5139951A (en) | Culture device having a detachable cell or tissue growth surface | |
| US5047347A (en) | Gas permeable culture flask and method for culturing mammalian cells | |
| US5462874A (en) | Dialyzed multiple well tissue culture plate | |
| GB2268187A (en) | Cell culture vessels | |
| JPS61108373A (en) | Cell culture apparatus and method | |
| EP0769048B1 (en) | Compartmentalized tissue culture flask | |
| GB2592812A (en) | Device for reduced oxygen concentration culture in microfluidic systems | |
| CN1249217C (en) | Cell culture incubator | |
| CN206279212U (en) | A kind of cell culture apparatus | |
| JPH11509420A (en) | Dialysis multiple well tissue culture plate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 361022 Xinguang Road 332, Haicang Xinyang Industrial Zone, Xiamen City, Fujian Province Patentee after: Yingke Xinchuang (Xiamen) Science and Technology Co.,Ltd. Address before: 361022 Xinguang Road 332, Haicang Xinyang Industrial Zone, Xiamen City, Fujian Province Patentee before: INTEC PRODUCTS Inc. (XIAMEN) |
|
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20060405 |