CN102337200A

CN102337200A - High-efficiency microcarrier cell-culture medium separating device

Info

Publication number: CN102337200A
Application number: CN2010102312230A
Authority: CN
Inventors: 回良杰
Original assignee: BEIJING XINYI YUANCHENG BIOLOGICAL TECHNOLOGY CO LTD
Current assignee: BEIJING XINYI YUANCHENG BIOLOGICAL TECHNOLOGY CO LTD
Priority date: 2010-07-20
Filing date: 2010-07-20
Publication date: 2012-02-01

Abstract

The invention relates to a perfusion device for high-density culture of an animal cell by adopting a microcarrier mode, in particular to a device for settling the microcarrier and separating the microcarrier from the culture medium.

Description

A kind of efficient microcarrier cell-substratum tripping device

Technical field

The perfusion device that adopts when the present invention relates to a kind of zooblast microcarrier mode high-density culture.Particularly, the present invention relates to a kind of microcarrier sedimentation and and the isolating device of substratum.

Background of invention

Since Van Wezel in 1967 uses microcarrier (micro-carrier) system to carry out the animal cell large-scale cultivation; Through nearly 50 years development; This technology gradually is gradually improved at present and is ripe; And be widely used in the cell engineering field to produce the cellular product that some have important practical and commercial value, like vaccine, gene engineering product etc.

The micro-carrier system culturing cell has significant advantage: the advantage that 1) has monolayer culture and suspension culture concurrently; 2) cell environment homogeneous of living in is that homogeneous phase is cultivated; 3) envrionment conditions (temperature, DO, pH etc.) is measured easily and is monitored; 4) has higher specific surface; 5) but cultivate operation systematize, robotization, reduced and polluted the chance that takes place.

Along with people's is to the reinforcement of disease prevention demand; Requirement to vaccine quality, output also improves thereupon; Enterprise wants meeting the market requirement simultaneously, improves a variety of causes such as profit, impels the method for new scale operation vaccine perfect constantly; The microcarrier cultured method is widely used, and is the development trend of large-scale production from now on.At present, external many biological products generally use bio-reactor to carry out large-scale industrial production, and domestic also have its launch products.These explain that all carrying out scale operation with microcarrier gets into the practical stage, cultivate to compare with traditional rolling bottle and can improve output, quality, reduce and pollute, and save manpower, reduce the advantages such as use of nutrient solution.

Training methods such as microcarrier perfusion help the high-density culture of cell, but are used for production of vaccine, should adopt stream to add formula and cultivate, and can fully improve the production of vaccine titre like this.Along with the continuous increase of microcarrier concentration, cell density also increases thereupon, obtain highdensity cell; The concentration of microcarrier can be suitably increased, but when microcarrier concentration surpasses 5g/L, the high-density growth of cell be kept; Just must take training method such as perfusion to guarantee the nutrition supply of cell; Otherwise because carrier concn is crossed conference and caused the nutrient solution nutrient consumption too fast, the meta-bolites accumulation is excessive, the cell state variation.So it is very important that perfusion device seems, and perfusion rate depends primarily on the velocity of separation of microcarrier-cell and substratum, the present invention has mainly carried out brand-new design on the microcarrier sedimenting system of tripping device.

Existing in the world microcarrier cell---substratum separation system is mainly divided two types: 1) the centrifugal cage assembly of screen membrane: this device utilizes the rotary sieve post; Separate microcarrier and substratum with cf-; This separation system advantage is that separation efficiency is high; Rotation drives with needing independently but need take big jar internal volume, and the low capacity jar (as≤14L) go up that stopping property is not easy to guarantee that filter screen is blocked easily when using; 2) gravity settling device, the main jar outer gravity vertical sedimentation system that utilizes of this device accomplishes sepn process in conjunction with recoiling device, and its shortcoming is when perfusion flow is big, and the microcarrier cell causes anoxic and nutritive deficiency after getting into sedimentation pipe easily; Need close observation,, need the artificial back-purge system of opening in sedimentation pipe when microcarrier accumulates when too much.The tripping device that the present invention adopts has overcome the filter screen obstruction and the microcarrier cell is accumulated too much shortcoming on filter screen, and does not need the special driving device, and cost of manufacture is low.

Summary of the invention

The object of the invention is to provide a kind of cell with microcarrier and upward growth thereof (being called for short the microcarrier cell) and the isolating device of substratum, comprises parts such as sedimentation chamber, anti-disturbance dividing plate, porthole (hole, pipe), pipe connecting, microcarrier filter screen and recoiling device.It is characterized in that sedimentation chamber forms the microvariations microcarrier cell gravity settling environment that meets the perfusion rate design, cooperate the substratum pump to accomplish sepn process.This apparatus features is that separation efficiency is high, and perfusion rate is apparently higher than the separation system of using on the market at present, and cheap, and the pair cell damage is little.When principle was slightly larger than substratum for utilizing microcarrier density, under the microvariations environment, the microcarrier cell can separate with substratum by the certain speed sedimentation.

Implementation method of the present invention is:

1, sedimentation chamber design sedimentation chamber is (ellipse) right cylinder or its distortion, is positioned at retort, and top seal also has the joint that is connected with pipe connecting, and the bottom is open, and inside is equipped with anti-disturbance dividing plate; Height of column is greater than 3cm, preferred 5～8cm; Cross-sectional area designs according to required perfusion flow.Require and resistant to elevated temperatures various material by meeting cell cultures, prepare like materials such as stainless steel, polyester, plastics.Sedimentation chamber design is main considers two factors: the 1) settling velocity of microcarrier in substratum, thus having determined the height of sedimentation chamber, experimental result shows that height can satisfy the requirement of most of kind microcarrier settling velocity at 3-9cm; 2) cross-sectional area depends on perfusion rate; Formula is: S=Vp ÷ (Vs * 1440) wherein S is the sedimentation chamber cross-sectional area, and unit is cm ²Vp is perfusion rate (ml/d); Vs be this part of microcarrier cell settlement speed (cm/min) can be single tube chamber form, also can parallelly connected a plurality of tube chambers, the UNICOM at the top.

2, it act as assistance sedimentation chamber formation microvariations liquid environment to prevent the disturbance baffle design, is beneficial to the settling process of microcarrier cell.It is the flap of installing along the cylinder direction, and lower end bending formation and horizontal plane form 45-80 ° of angle, are preferably 60 °, and this angle design should not make the microcarrier cell be trapped on the dividing plate;

3, porthole (hole, pipe) design is made up of the sealing-duct of transparent material preparation, and vertical the placement is connected in the sedimentation chamber upper pipeline, and its internal diameter is identical with pipe connecting or big slightly.The observation tube outer wall can be installed photophore/optical sensor, is used to detect microcarrier and builds up, and activates recoiling device.

4, filter screen design microcarrier filter screen is the net of aperture less than the microcarrier diameter; Material is stainless steel or health ranks such as nylon, polyester; Be installed in the top of porthole (hole, pipe); Be used for collecting the microcarrier cell of escaping from from sedimentation chamber, and in being accumulated to when a certain amount of with recoiling device refunds jar.

5, pipe connecting design pipe connecting inserts the opening and the sealing of tank body top cover for connecting the pipeline of sedimentation chamber and observation tube.

6, recoiling device design recoiling device is made up of a threeway and corresponding pipeline, liquor pump, act as when depositing too much microcarrier cell on the filter screen, utilizes reverse flow in the settling refunds retort.

Description of drawings

Fig. 1 sedimentation chamber design diagram

A: sedimentation chamber and pipe connecting interface

B: sedimentation chamber

C: dividing plate

Fig. 2 observation tube and filter screen synoptic diagram

A: interface on the observation tube has sealing thread and observation tube to form tightness system

B: elastomeric pad

C: filter screen

D: observation tube

E: photodiode

F: observation tube lower interface

G: sight sensor provides the microcarrier cell to build up information to system

Fig. 3 recoiling device synoptic diagram

A: infusion pump, normal perfusion is used

B: recoil pump is used in the microcarrier cell refunds jar

C: the pipeline that connects the observation tube upper end

D: threeway

Specific embodiment

Following instance is that the present invention further is detailed, but content of the present invention is not to be defined in this.

1, perfusion flow is the sedimentation chamber design of 20000ml/d: according to surveying and determination; When using the CYTODEX-1 microcarrier; Settling velocity is about 1.2cm/min, according to formula, and design (ellipse) right cylinder cross-sectional area S=Vp ÷ (Vs * 1440)=20000 ÷ (1.2 * 1440)=11.57cm ², 2～3 dividing plates of set inside.

2, perfusion detects: in capacity is the cell response jar of 14L, adds microcarrier according to 15g/L, and carry out the VERO cell cultures, regulating infusion pump speed is 15ml/min, observes through 24h, does not find that microcarrier builds up in observation tube, and perfusion flow is 21.6L/d.

Claims

1. a microcarrier cell---substratum tripping device is partly formed comprising sedimentation chamber, anti-disturbance dividing plate, porthole (hole, pipe), pipe connecting, microcarrier filter screen and recoiling device etc.It is characterized in that sedimentation chamber forms microvariations microcarrier (cell) the gravity settling environment that meets the perfusion rate design, cooperate the substratum pump to accomplish sepn process.

2. sedimentation chamber according to claim 1 is (ellipse) right cylinder or its distortion, is positioned at retort, and top seal also has the joint that is connected with pipe connecting, and the bottom is open, and inside is equipped with anti-disturbance dividing plate; Height of column is greater than 3cm, preferred 5～8cm; Cross-sectional area designs according to required perfusion flow.Require and resistant to elevated temperatures various material by meeting cell cultures, prepare like materials such as stainless steel, polyester, plastics.

3. anti-disturbance dividing plate according to claim 1, it act as assists sedimentation chamber to form the microvariations liquid environment, is beneficial to the settling process of microcarrier cell.It is the flap of installing along the cylinder direction, and lower end bending formation and horizontal plane form 45-80 ° of angle and be preferably 60 °.

4. porthole according to claim 1 (hole, pipe) is used to observe or the separating effect of microcarrier for jar outside, and by the sealing-duct of transparent material preparation, the vertical placement is connected in the sedimentation chamber upper pipeline, and its internal diameter is identical with pipe connecting or big slightly; The observation tube outer wall can be installed photophore/optical sensor, is used to detect the microcarrier deposition, activates recoiling device.

5. pipe connecting according to claim 1 inserts the opening and the sealing of tank body top cover for connecting the pipeline of sedimentation chamber and observation tube.

6. microcarrier filter screen according to claim 1 is the net of aperture less than the microcarrier diameter, and material is stainless steel or health ranks such as nylon, polyester, is installed in the top of porthole (hole, pipe).

7. recoiling device according to claim 1 is made up of a threeway and corresponding pipeline, liquor pump, act as when depositing too much microcarrier cell on the filter screen, utilizes reverse flow in the settling refunds retort.

8. tripping device according to claim 1, perfusion rate can reach 1-10 times (1-10V/d) of retort volume every day when it was used for cell cultures.