CN1125173C - Three-dimensional cell and tissue culturing device with two rotating shafts - Google Patents
Three-dimensional cell and tissue culturing device with two rotating shafts Download PDFInfo
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- CN1125173C CN1125173C CN 99119640 CN99119640A CN1125173C CN 1125173 C CN1125173 C CN 1125173C CN 99119640 CN99119640 CN 99119640 CN 99119640 A CN99119640 A CN 99119640A CN 1125173 C CN1125173 C CN 1125173C
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Abstract
The present invention relates to a double-shaft rotating three-dimensional cell and tissue culture apparatus in tissue engineering, which is mainly composed of an inner cylinder culture liquid flowing chamber, a cell culture chamber and a culture apparatus body with an integral structure of three sheathed cylinders, wherein the outer side of the culture apparatus body is provided with a gas chamber, and the culture apparatus body is arranged on a transversal rotating frame rotating with 2 freedom degrees. The gas chamber and the culture liquid flowing chamber are respectively driven by a rotating speed adjusting mechanism and a motor through a shaft, and make differential rotation after coaxial rotation. End effect is eliminated by swinging around a transverse shaft at a low speed, and a good environment for the three-dimensional growth of cells, cell aggregates and tissues is built. Simultaneously, the present invention is favorable for culturing indoor oxygen and transporting CO2 nutrients and metabolite.
Description
The device that the present invention relates to use in biomedical engineering is especially for the twin shaft rotary cell/tissue dimensional culture device in forward position-organizational project (Tissue Engineering) field of biomedical engineering.
' organizational project ' speech is to put forward in the academic discussion that American National Natural Science Fund (NSF) is held in August, 1987.A work group by NSF had done to define to its intension in 1988, that is: " principle of applied engineering science and life science and method; explain the structure of mammalian tissues---functional relationship; and the development have active worker's surrogate, recover, keep or improve the function of tissue ".Its core is that living tissue or other biological substitution thing of having grown and having had specific function are applied to clinical by the isolated culture of Mammals (comprise the people, and even patient self) cell.The problem in science that organizational project relates to is a lot, and cell/tissue dimensional culture device is one of its gordian technique.The difference that used bio-reactor and traditional cell culture processes have essence in it and the modern biochemistry engineering (as fermentation engineering, pharmaceutical industry etc.).As used bio-reactor such as fermentation industry, pharmacy industry by means of mechanical stirring (comprise the laboratory uses shake bottle, shaking table etc.) or be blown into gas and cause that methods such as forced convection avoid the culture sedimentation, and improve nutrition supply, this is successful in microorganism culturing (as production of antibiotics), is unfavorable but cultivate for mammalian cell.Because: (1) mammalian cell cultivate than microorganism ' tender and lovely ' many, the mobile shearing that mechanical stirring causes and directly effect be easy to damage and even destroy cell, cause necrosis.(2) the mobile generation of cutting that causes of mechanical stirring makes cell be difficult to (even can not) to assemble, and cell aggregation is the first step that becomes tissue from cell cultures.
In addition, the conventional cell cultural method is before the mid-90, and medical circle fails to turn out to be had with next in the same or analogous biological tissue of body group function.Its major cause is: inevitably action of gravity makes cell (or microcarrier) fall to the incubator bottom, the contact inhibition of cell between the incubator wall makes institute's cultured cells true three-dimensional not grow, thereby limited the cell function differentiation, length does not become tissues needed.And the space microgravity environment to be cell three-dimensional growth created condition.The development space bio-reactor is just set about from the mid-80 by NASA (NASA).At the initial stage nineties, NASA Johnson has obtained major progress in the center in this respect, has developed Rotary pipe type bio-reactor (RWV).And nineteen ninety-five space shuttle carry in the experiment and successfully turn out colon cancer tissue, tissue slice is observed and is shown: it have with in similar mechanism of body tissue.And its size is bigger 30 times than what cultivate with traditional method, reaches 2 centimetres.Carried out the articular cartilage tissue culture experiment again in 1997, successfully turned out with the body cartilaginous tissue similar and save cartilaginous tissue, be of a size of a centimetre magnitude.When this novel bio-reactor moves on ground, also can avoid gravity settling, thereby guarantee the cell three-dimensional growth by nutrient solution mobile hydromeehanics effect in the reactor.As reference: [1] Culturing a Future, Fall 1998, Microgravity News but this novel bio-reactor also is imperfect, the pressure stress level can not be regulated and control in the first culturing room; Its two be incubator when using in fact suspended substance be three-dimensional in the indoor motion of cell cultures, but it will and cause the cell/tissue dimensional culture totally unfavorable to the convergence of inner core surface near the end time; Bring the end effect problem that is unfavorable for the cell three-dimensional growth, as shown in Figure 1.D=6cm among the figure, d=1.5cm, L=6cm; N1=5rad/s, N2=1.25rad/s, k=0.3; Starting point: X=1, Y=2.0cm, Z=0.2cm..
The objective of the invention is to overcome the shortcoming of prior art, in order to solve the end effect problem that is unfavorable for the cell three-dimensional growth that occurs when existing incubator uses, i.e. the culture settlement issues that causes by the finite length end effect; With in order to realize that the pressure stress level can be regulated and control in the culturing room, thereby provide the hydrodynamic force that motion of viscous produces between a kind of two cylinders of using coaxial rotation to overcome gravity settling, make culture (or microcarrier or cell aggregation etc.) be in three-dimensional suspended state, not only can do coaxial rotation but also can do on the basis of differential rotation at the two cylinders of incubator, also can do side throw, to obtain the twin shaft rotary cell/tissue dimensional culture device of best three dimensional growth condition.
The object of the present invention is achieved like this: twin shaft rotary cell/tissue dimensional culture device provided by the invention comprises that concentric locking collar puts the nutrient solution stream of putting
Moving chamber, cell culture chamber and air chamber, the nutrient solution flow chamber is an inner core, is the hollow out round tube type bracing frame that fixedly is covered with liquid/liquid exchange membrane on its outside surface; Cell culture chamber is an intermediate cylinder, is the hollow out round tube type bracing frame that fixedly is covered with the liquid/gas exchange membrane on its outside surface; The same end of nutrient solution flow chamber and cell culture chamber is separately installed with rotatable end axle, and air chamber is a urceolus, and maintains static, and the inwall of air chamber is the outer wall of cell culture chamber; Two groups of rotating mechanisms of being made up of motor and variator are connected with the end axle of nutrient solution flow chamber and cell culture chamber respectively, and these two groups of rotating mechanisms of being made up of motor-variator are fixed on the support of base; On the nutrient solution flow chamber end axle separation mechanism is housed, the outlet of nutrient solution import and nutrient solution is arranged on it, the liquid pipe of the infusion pump in the liquid-supplying system links to each other with the nutrient solution memory, after passing the exit deoscillator earlier by the perforated tube pipeline again, enter cell culture chamber to the cell culture chamber terminal, the output channel of cell culture chamber is connected in the nutrient solution dialyzer mutually, and it communicates with nutrient solution withdrawer and meta-bolites collector respectively; With on the tracheae that source of the gas and air inlet of air chamber are connected voltage stabilizing/relief valve is installed, the air outlet of air chamber is connected on the residual air collector by vapor pipe, comprise that also the side throw support is fixed on the whole bearing, another motor is installed in side throw support inboard, and the axle of this motor passes the side throw support and another slide bar B is fixed together; By two side throw rotating shafts on the lateral rotation framework inboard incubator main body is fixed, fix a slide bar A in the side throw rotating shaft again; Two slide bars slide by the groove on it and match, and side throw amplitude modulation, amplitude limit parts are passed a slide bar A and be fixed on slide bar B; Drive slide bar by the lateral rotation torque motor and do the side throw campaign, wherein side throw amplitude modulation is between 8 °-20 °; The nutrient solution flow chamber is connected with axle, and axle is connected with a motor by rotating speed adjusting mechanism; Cell culture chamber is connected with another axle, and is connected with another motor by rotating speed adjusting mechanism, and inlet mouth is connected with the tracheae of airing system on the air chamber; A nutrient solution import and an outlet are connected with import and export on the feed flow transport path respectively; Described amplitude limit parts can be bolt.
The nutrient solution flow chamber is that (radius of inner core is R to inner core
1), cell culture chamber is that (radius of intermediate cylinder is R to intermediate cylinder
2), air chamber is that (radius of urceolus is R to urceolus
3), wall of the outer-rotor is fixed; Two groups of rotating mechanisms of being made up of motor and variator of controller coupling regulation and control are connected with the axle of inner core and intermediate cylinder respectively.Motor is fixed on the support of base, and the rotating mechanism of two groups of motor-variator compositions makes inner core and intermediate cylinder both can rotate (ω synchronously
1=ω
2), also can differentially rotate (ω
1≠ ω
2), (ω
3=0).ω
1(t)=ω
2(t) time, cylinder R
1, R
2Rotation synchronously; ω
1(t) ≠ ω
2(t) time, nutrient solution flow chamber (inner cylinder R
1), cell culture chamber (intermediate cylinder R
2) differential rotation.On the inner core end axle separating component is housed, the outlet of nutrient solution import and nutrient solution is arranged on it, its effect is: (1) separates nutrient solution import and export and rotation inner core axle, does not rotate thereupon; (2) keep apart flowing to fresh medium and flowing out nutrient solution.
(1) at cell culture chamber middle part, condition that culture suspends is mainly by angular velocity of rotation (ω in process of growth
1=ω
2During=ω), decision such as liquid phase medium viscosity, culture and nutrient solution density difference in the culturing room.Along with culture yardstick (d represents with equivalent spherical diameter) increases, also increase for avoiding the needed circular frequency of gravity settling.Fig. 2 is that culture yardstick and suspension culture minimum speed concern example.
(2) in fact the motion of suspended substance is three-dimensional, and as shown in Figure 1, it will be to the convergence of inner core surface near the end time, and this is disadvantageous for the cell/tissue dimensional culture, and is especially true when the culture yardstick is big.The present invention is in order to overcome cell and to grow in the dimensional culture device, and it will be to the convergence of inner core surface near the incubator end time, cause culture sink to the incubator bottom bring unfavorable.The present invention be when grown cell culture to be placed on cell cultures indoor, by liquid-supplying system and airing system to nutrient solution flow chamber feed flow with to the air chamber air feed, exchange by liquid/liquid exchange membrane or gas/liquid exchange membrane, culture provides oxygen and necessary nutrition in the pair cell culturing room, two cylinders in the incubator main body are under the drive of two motors simultaneously, coaxially rotate at first then and doing differential rotation, make cell in culturing room's inner suspension growth, the stress distribution in the culturing room also is regulatable; Because the axle of many side throws, twin shaft rotates, and has increased a regulation and control factor, helps the regulation and control of transport of substances and mechanical environment in the culturing room widely.But the installation site of side throw rotating shaft must by with the barycenter (center of gravity) of air chamber/culturing room/training liquid flow chamber integral structure and rotating frame lateral rotation platform, swing rate could be evenly.In addition, the amplitude of side throw is advisable with 8 °-20 °.
Effect of the present invention and advantage
But the configuration of cell culture chamber, air chamber and the nutrient solution flow chamber of twin shaft rotary cell/tissue dimensional culture device provided by the invention is synchronous variable-speed both, also can differentially regulate, and the stress distribution in the culturing room is regulatable; Because twin shaft rotation, by low speed swing around transverse axis, eliminate end effect, can significantly improve the culture that end effect causes is inclined to inner tubal wall face accumulative, increased a regulation and control factor, help the regulation and control of transport of substances and mechanical environment in the culturing room, build an environment that more helps cell, cell aggregation and tissue three-dimensional growth.Simultaneously, more help oxygen in the culturing room, CO
2Transporting of nutrition and metabolite, and increased an approach for the regulation and control of culturing room's interior mechanics environment.
Below in conjunction with embodiment and accompanying drawing the present invention is described in detail:
Accompanying drawing 1 is the end effect figure that the incubator finite length causes
Accompanying drawing 2 is incubator culture size of the present invention (d) and the required minimum speed of suspension culture
Accompanying drawing 3 is twin axle rotary cell/tissue dimensional culture device structural representation circles of the present invention
Accompanying drawing 4 is twin axle rotary cell/tissue dimensional culture device feed flow transport path synoptic diagram of the present invention
Accompanying drawing 5 is twin axle rotary cell/tissue dimensional culture device air feed transport path synoptic diagram of the present invention
Embodiment 1
Press the twin axle rotary cell/tissue dimensional culture device that Fig. 3-5 makes playscript with stage directions invention, 1 this dimensional culture device comprises a nutrient solution flow chamber (3)/cell culture chamber (1)/(7) three concentric(al) circles cylinders of air chamber in the drawings, the integrated incubator agent structure that cover is dressed up, the incubator agent structure is made cylinder with polycarbonate material, and commercially available liquid/liquid exchange membrane or the gas/liquid exchange membrane of usefulness between three concentric(al) circles cylinders separates; Motor (11) is fixed on lateral rotation framework (38) one ends, the axle of motor (11) is by gear (10) and gear (14) interlock, the axle of nutrient solution flow chamber (3) passes outside the lateral rotation framework (38), this axle latter end in framework (38) has a gear (5), this axle exposes the outer section of lateral rotation framework (38) and goes up the split tunnel (6) that cover one polycarbonate material is made, it is communicated with nutrient solution flow chamber (3), split tunnel has a fluid inlet on (6), (14) and a liquid outlet (15), split tunnel is equipped with a sealing-ring in (6), isolates fresh medium and effusive nutrient solution; Incubator main body two ends are fixed on the stainless lateral rotation framework (38), and lateral rotation framework (38) is fixed on the side throw support (34), and side throw support (34) is positioned on whole bearing (35); Respectively incubator main body three is fixed together in lateral rotation framework (38) both sides by a side throw rotating shaft (30), i.e. the installation site of side throw rotating shaft must by with the barycenter (center of gravity) of air chamber/culturing room/training liquid flow chamber integral structure and rotating frame lateral rotation platform (7); Fix a slide bar B (37) in the side throw rotating shaft (30) again, side throw support (34) inboard is fixed a motor (33) and is linked to each other with horizontal balance staff (30), and the axle of motor (33) passes side throw support (34) and is fixed together with slide bar (36); On slide bar B (36) and the slide bar B (37) groove is arranged, both match in slip, and a bolt (31) passes slide bar and upward fixing with slide bar B (36), but bolt (31) can slide in the groove of slide bar B (37).
(6) split tunnel of making of polycarbonate;
(38) be the lateral rotation framework of stainless material;
(8), (9) are the rotating speed adjusting mechanism that cell cultures indoor and outdoor barrel connects;
(10), (11) are CD-ROM drive motor, link to each other with rotating speed adjusting mechanism (8), (9) respectively;
(12), (13) and (14), (15) are respectively gas import and export on the air chamber (7) and the nutrient solution import and export on the nutrient solution flow chamber (3), be connected with nutrient solution feed flow transport path with the air feed part respectively;
(30) be the side throw rotating shaft, the installation site of side throw rotating shaft must by with the barycenter (center of gravity) of air chamber/culturing room/training liquid flow chamber integral structure and rotating frame lateral rotation platform (7); ,
(31) be to do side throw amplitude modulation, amplitude limit parts with a bolt,
(32) be horizontal balance staff;
(33) be the lateral rotation torque motor, be installed in support frame (34) inboard, do not see among the figure;
(34) be the side throw support, lateral rotation framework (38) is fixed on it by side throw rotating shaft (30);
(35) be whole bearing, above-mentioned (30)-(35) all make of stainless steel;
(36) slide bar A;
(37) slide bar B; Use the mating parts of two band chutes of stainless steel making, slide by chute between the two and adjust the intersecting angle of two parts, it is the amplitude of side throw, pin slide bar A with bolt (31) after adjusting intersecting angle, in slide bar B (36) groove, slide but bolt (31) can drive slide bar A (36).Be equipped with a swing with horizontal balance staff (32) at the end of slide bar B (36), swing by one at the end of slide bar A (37) and be fixed on the side throw support (34) with horizontal balance staff (30).
The nutrient solution transport system of present embodiment, as shown in Figure 4, in the drawings:
(18) be the fresh medium memory;
(19) be the infusion pump of flow, adjustable in pressure;
(20) for input nutrient solution void channels, enter culturing room to culturing room terminal through the outlet section deoscillator.
(21) the outlet deoscillator is the commercially available hose clamp that has adjustment screw; In order to regulate the nutrient solution flow of input culturing room, reach and regulate the internal pressure (P of culturing room
b(ζ));
(22) be the nutrient solution dialyzer;
(23) be the nutrient solution withdrawer;
(24) be the meta-bolites collector; Wherein the liquid pipe of infusion pump (19) links to each other with nutrient solution memory (18), after a end by void channels passes outlet deoscillator (21) earlier again, enter cell culture chamber to culturing room's terminal, the output channel of the cell culture chamber nutrient solution dialyzer (22) that links to each other, it communicates with nutrient solution withdrawer (23) and meta-bolites collector (24) respectively; Wherein air inlet of air chamber and source of the gas (25) are by the voltage stabilizing on the tracheae, and relief valve (26) connects; The air chamber air outlet connects on the residual air collector (29) by vapor pipe (28).
The air feed of present embodiment---exhaust system, as shown in Figure 4, in the drawings:
(25) be source of the gas, common bomb;
(26) for voltage stabilizing, relief valve, by to voltage stabilizing, the regulating and controlling effect of relief valve reaches the purpose of regulating culturing room's internal stress;
(27) be inlet pipe (linking to each other) with figure-6 they (9);
(28) be vapor pipe (linking to each other) with figure-6 they (10);
(29) be the residual air collector; Wherein air inlet of air chamber and source of the gas (25) be by the voltage stabilizing on the tracheae, relief valve (26)
Connect; The air chamber air outlet connects on the residual air collector (29) by vapor pipe (28).
Claims (2)
1, a kind of twin shaft rotary cell/tissue dimensional culture device, it is characterized in that: comprise that concentric locking collar puts nutrient solution flow chamber, cell culture chamber and the air chamber of putting, the nutrient solution flow chamber is an inner core, is the hollow out round tube type bracing frame that fixedly is covered with liquid/liquid exchange membrane on its outside surface; Cell culture chamber is an intermediate cylinder, is the hollow out round tube type bracing frame that fixedly is covered with the liquid/gas exchange membrane on its outside surface; The same end of nutrient solution flow chamber and cell culture chamber is separately installed with rotatable end axle, and air chamber is for maintaining static urceolus, and its inwall is the outer wall of cell culture chamber; Two groups of rotating mechanisms of being made up of motor and variator are connected with the end axle of nutrient solution flow chamber and cell culture chamber respectively; Be fixed on the support of base with these two groups of rotating mechanisms of forming by motor-variator that the nutrient solution flow chamber links to each other with cell culture chamber respectively; On the nutrient solution flow chamber end axle separation mechanism is housed, the outlet of nutrient solution import and nutrient solution is arranged on it, the liquid pipe of the infusion pump in the liquid-supplying system links to each other with the nutrient solution memory, after passing the exit deoscillator earlier by the perforated tube pipeline again, enter cell culture chamber to the cell culture chamber terminal, the output channel of cell culture chamber is connected in the nutrient solution dialyzer mutually, and it communicates with nutrient solution withdrawer and meta-bolites collector respectively; With on the tracheae that source of the gas and air inlet of air chamber are connected voltage stabilizing/relief valve is installed, the air outlet of air chamber is connected on the residual air collector by vapor pipe, comprise that also the side throw support is fixed on the whole bearing, another motor is installed in side throw support inboard, and the axle of this motor passes the side throw support and another slide bar B is fixed together; By two side throw rotating shafts on the lateral rotation framework inboard incubator main body is fixed, fix a slide bar A in the side throw rotating shaft again; Two slide bars slide by the groove on it and match, and side throw amplitude modulation, amplitude limit parts are passed a slide bar A and be fixed on slide bar B; Drive slide bar by the lateral rotation torque motor and do the side throw campaign, wherein side throw amplitude modulation is between 8 °-20 °; The nutrient solution flow chamber is connected with axle, and axle is connected with a motor by rotating speed adjusting mechanism; Cell culture chamber is connected with another axle, and is connected with another motor by rotating speed adjusting mechanism, and inlet mouth is connected with the tracheae of airing system on the air chamber; A nutrient solution import and an outlet are connected with import and export on the feed flow transport path respectively.
2, by the described twin shaft rotary cell/tissue of claim 1 dimensional culture device, it is characterized in that described amplitude limit parts is a bolt.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99119640 CN1125173C (en) | 1999-09-23 | 1999-09-23 | Three-dimensional cell and tissue culturing device with two rotating shafts |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99119640 CN1125173C (en) | 1999-09-23 | 1999-09-23 | Three-dimensional cell and tissue culturing device with two rotating shafts |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1252437A CN1252437A (en) | 2000-05-10 |
| CN1125173C true CN1125173C (en) | 2003-10-22 |
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ID=5281005
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99119640 Expired - Fee Related CN1125173C (en) | 1999-09-23 | 1999-09-23 | Three-dimensional cell and tissue culturing device with two rotating shafts |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1125173C (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100357425C (en) * | 2005-03-16 | 2007-12-26 | 中国科学院空间科学与应用研究中心 | Rotator able to be micro observed |
| CN1304566C (en) * | 2005-04-11 | 2007-03-14 | 清华大学 | Complex organ-like precursor with culture device and structure thereof and culturing method |
| CN101560469B (en) * | 2008-04-14 | 2012-10-31 | 上海交通大学医学院附属上海儿童医学中心 | Rotary dual-chamber stem cell synchronous augmentation and division inoculator |
| CN102007515B (en) * | 2008-04-18 | 2013-06-12 | 皇家飞利浦电子股份有限公司 | Segmenting pulmonary arteries |
| CN102086438B (en) * | 2009-12-07 | 2012-12-19 | 韩春茂 | Device and method for biological culture of cell or tissue engineering |
| CN103789208A (en) * | 2014-02-19 | 2014-05-14 | 上海交通大学医学院附属第九人民医院 | Biaxial rotary bioreactor |
| CN104031835B (en) * | 2014-05-21 | 2015-11-25 | 中国人民解放军第四军医大学 | The replaceable nutrient solution cell micro-gravity system of three-dimensional revolution |
| US20210331956A1 (en) * | 2016-09-19 | 2021-10-28 | Linde Aktiengesellschaft | Methods for wastewater treatment |
| CN110157617B (en) * | 2019-04-29 | 2021-07-16 | 重庆大学附属肿瘤医院 | Cervical cancer cell three-dimensional culture device and application thereof |
| CN114350519B (en) * | 2022-03-15 | 2022-05-31 | 广州加泰医药科技有限公司 | Cell incubator for bioreaction device |
-
1999
- 1999-09-23 CN CN 99119640 patent/CN1125173C/en not_active Expired - Fee Related
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|---|---|
| CN1252437A (en) | 2000-05-10 |
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