CN108060081A - A kind of multi-layer planar filling type bioreactor - Google Patents

Abstract

The invention belongs to the technical field of bioreactors, and specifically relates to a multi-layer plate perfusion bioreactor, which includes a perfusion cavity and a culture mechanism composed of multi-layer plates arranged in the perfusion cavity. There are a culture solution inlet pipe and a culture solution discharge pipe, the perfusion chamber is divided into a culture chamber and a buffer chamber, the culture mechanism is set in the culture chamber, and the outlet of the culture solution inlet pipe extends to Below the middle part of the buffer chamber, the upper part of the buffer chamber communicates with the culture chamber. The purpose of the present invention is to provide a bioreactor that can be used for large-scale in vitro culture of cells and provide a more uniform flow field shear force environment for cell culture.

Description

A multi-layer plate perfusion bioreactor

technical field

The invention relates to the technical field of bioreactors, in particular to a multi-layer plate perfusion bioreactor.

Background technique

A bioreactor is an in vitro device used to simulate organisms to provide suitable nutrients, physical environment and chemical environment for cell growth. Bioreactors have shown great application potential in drug production, tissue engineering, and regenerative medicine.

The flow of the culture solution in the bioreactor can make the substances in the culture solution more evenly distributed, and facilitate the cells in the reactor to obtain nutrients and discharge metabolic waste. At present, there are many types of bioreactors, which can be divided into hybrid bioreactors and perfusion bioreactors according to the flow mode of the culture medium. Among them, the perfusion bioreactor has the advantages of stable flow field and high mass transfer efficiency, and has received more and more attention. Much attention. Perfusion bioreactors can be divided into hollow fiber reactors, fluidized bed reactors and multi-layer plate reactors according to different configurations. Among them, the multi-layer plate perfusion bioreactor uses multi-layer plates arranged in parallel as cell carriers. The culture medium flows between the plates and converges to the outlet of the reactor or flows into the next layer of plates through the diversion holes. It has been applied to Various clinical experiments such as bioartificial liver and stem cell culture in vitro.

Chinese patent ZL 201310197609.8 discloses a perfusion bioreactor with a double-layer circulation system and its application method, including a culture tank, an outer circulation system outside the culture tank, an inner circulation system inside the culture tank and gas Filling system; solve the contradictory demand for the circulation of the culture medium during the culture process of cells, and can effectively improve the solubility of oxygen in the culture medium and the uniformity of distribution, which is conducive to the discharge of waste gas in the culture medium, and can effectively eliminate Air bubbles enter the cell culture area.

Studies have shown that the shear force of the flow field in the reactor has a huge impact on the growth of cells. An appropriate shear force environment can increase cell activity or promote cell differentiation, and an excessive shear force environment will inhibit cell activity and even cause cell apoptosis. . Perfusion bioreactors have high flow rates at the perfusion inlet and often have areas of high shear. In addition, large shear force will be generated when the bubbles burst, which is not conducive to the growth of cells. In the existing multi-layer plate bioreactor, the culture medium is directly poured into the multi-layer plate structure, which easily causes uneven distribution of shear force and is not conducive to cell growth.

Contents of the invention

The object of the present invention is to provide a kind of multi-layer flat plate perfusion bioreactor, this reactor overcomes the high shear force that existing multi-layer multi-layer flat plate perfusion bioreactor exists at the perfusion inlet, can be used for cell large-scale in vitro Culture, to provide a more uniform flow field shear force environment for cell culture.

In order to achieve the above object, the present invention provides the following technical solutions: a multi-layer plate perfusion bioreactor, including a perfusion cavity and a culture mechanism composed of multi-layer plates arranged in the perfusion cavity, the perfusion cavity A culture solution inlet pipe and a culture solution discharge pipe are provided, the perfusion chamber is divided into a culture chamber and a buffer chamber, the culture mechanism is set in the culture chamber, and the outlet of the culture solution inlet pipe extends Below the middle part of the buffer chamber, the upper part of the buffer chamber communicates with the culture chamber.

In the above-mentioned multi-layer flat plate perfusion bioreactor, the perfusion cavity is provided with a conduit communicating with the atmosphere.

In the above-mentioned multi-layer flat plate perfusion bioreactor, the culture mechanism includes a central guide tube and a first fixed frame and a second fixed frame that can be opened and closed. A plurality of first culture plates are detachably fixed from top to bottom, and a plurality of second culture plates corresponding to the first culture plates are detachably fixed on the second fixing frame from top to bottom , the first culture plate is provided with a first recess, and the second culture plate is provided with a second recess;

When the first fixing frame and the second fixing frame are closed, the first concave portion of the first culture plate and the second concave portion of the second culture plate form a ring that cooperates with the central draft tube;

The central guiding tube is provided with a through hole communicating with the culture chamber, and the culture solution draining tube is connected to the bottom of the central guiding tube.

In the above-mentioned multi-layer flat plate perfusion bioreactor, one end of the first fixing frame is hingedly connected to one end of the second fixing frame.

In the above-mentioned multi-layer flat plate perfusion bioreactor, the first fixing frame is provided with a first engaging part for engaging the first culture plate, and the second fixing frame is provided with a locking part for engaging the first culture plate. The second engaging part of the second culture plate; the central guide tube is provided with a third engaging part for engaging with the first concave part and the second concave part.

In the above-mentioned multi-layer plate perfusion bioreactor, the first culture plate and the second culture plate are semicircular.

In the above-mentioned multi-layer flat plate perfusion bioreactor, the other end of the first fixing frame and the other end of the second fixing frame are fixedly connected by a snap-fit rack.

In the above-mentioned multi-layer flat plate perfusion bioreactor, an observation mechanism is also included, and the observation mechanism is arranged on the perfusion cavity and directly above the cultivation mechanism.

In the above-mentioned multi-layer flat plate perfusion bioreactor, the observation mechanism includes a watertight observation window arranged on the perfusion cavity and an imaging device detachably arranged above the watertight observation window; the imaging device There is an auxiliary light source on it.

In the above-mentioned multi-layer plate perfusion bioreactor, the multi-layer plate of the culture mechanism is a multi-layer plate of high light-transmitting material.

Compared with prior art, the beneficial effect of the present invention is:

The perfusion cavity of the multi-layer flat bioreactor of the present invention introduces a buffer cavity, which eliminates the adverse effects of the high shear force caused by the perfusion of the culture fluid on the cells, provides a good microenvironment for the growth of the cells, and is conducive to cell adhesion, grow. The culture mechanism of the present invention takes into account the low disassembly efficiency of the cell carrier in the bioreactor when the cells are harvested, and greatly improves the disassembly efficiency by introducing the first fixed frame and the second fixed frame that can be opened and closed. , which shortens the time that the cell plate carrier is exposed to the outside world, thereby reducing the probability of sample contamination during the process. The observation mechanism of the present invention can observe the growth state of cells on the culture plate in real time, thereby providing a basis for timing selection of operations such as cell harvesting, and the imaging device can be installed and taken out at any time, which is convenient for disinfection and replacement of equipment at any time.

Description of drawings

Fig. 1 is the sectional view of embodiment 1 of the present invention;

Fig. 2 is the perspective view of the cultivation mechanism of embodiment 1 of the present invention;

3 is a three-dimensional expanded view of the culture mechanism of Embodiment 1 of the present invention;

Fig. 4 is the sectional view of the observing mechanism of embodiment 1 of the present invention;

Fig. 5 is a partial sectional view of Embodiment 1 of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

Please refer to Figures 1-5, a multi-layer plate perfusion bioreactor, including a perfusion chamber 1 and a culture mechanism 2 composed of multi-layer plates arranged in the perfusion chamber 1, on the perfusion chamber 1 A culture solution inlet pipe 11 and a culture solution discharge pipe 12 are provided, the perfusion chamber 1 is divided into a culture chamber 13 and a buffer chamber 14, the culture mechanism 2 is arranged in the culture chamber 13, and the culture The outlet of the liquid inlet pipe 11 extends below the middle of the buffer chamber 14, the upper part of the buffer chamber 14 communicates with the culture chamber 13, and the perfusion chamber 1 is provided with a conduit communicating with the atmosphere.

During use, the culture solution fluid is injected into the middle or lower part of the buffer chamber 14 through the culture solution inlet pipe 11. After being buffered by the buffer chamber 14, the fluid shear force becomes weaker and enters the culture chamber 13, which is beneficial to the activity of the cells. maintenance and cell differentiation.

It has been proved by experiments that the multi-layer plate is cleaned and installed on the plate support to form the culture mechanism 2, and the culture mechanism 2 with the multi-layer plate structure is soaked in 75% alcohol solution, then taken out and placed in an ultraviolet environment to air dry. After cleaning the shell of the reactor, put it into an autoclave for sterilization, and assemble the culture mechanism 2 with a multi-layer plate structure into the reactor on the ultra-clean bench, and place the whole reactor in 5% CO ₂ at 37°C in the cell culture incubator. Take human bone marrow mesenchymal stem cells and place them in culture flasks to grow to 10 million cells. After the cells are digested, a cell suspension is made and pumped into the reactor through a peristaltic pump. After standing still for 24 hours, after the cells adhered to the plate, the reactor was connected to the BioBundles fermenter through a silicone tube to adjust the parameters of the culture medium and circulate perfusion. After culturing for 10 days, the cell morphology was observed in real time through the observation device, and the cells reached 80% confluence. The multi-layer plate was taken out on the ultra-clean bench, and the number of cells was counted to reach 120 million, realizing the rapid expansion of stem cells in vitro. As a control, a reactor chamber without a buffer chamber was used for stem cell expansion experiments in vitro, and the same culture mechanism 2 was used and an equal amount of stem cell suspension was injected. After culturing for 10 days, the plate was taken out for cell counting, and the number of cells reached 82 million. This shows that the buffer chamber can effectively reduce the adverse effect of the flow field environment on cell growth and promote the rapid expansion of cells in vitro.

In the present embodiment, the cultivation mechanism 2 includes a central guide tube 21 and a first fixing frame 22 and a second fixing frame 23 that can be opened and closed. A plurality of first culture plates 24 are detachably fixed below, and a plurality of second culture plates 24 corresponding to the first culture plates 24 are detachably fixed on the second fixing frame 23 from top to bottom. Plate 25, the first culture plate 24 is provided with a first recess 26, and the second culture plate 25 is provided with a second recess 27;

When the first fixing frame 22 and the second fixing frame 23 are closed, the first recess 26 of the first culture plate 24 and the second recess 27 of the second culture plate 25 form a ring that cooperates with the central draft tube 21;

The central guide tube 21 is provided with a through hole 28 communicating with the culture chamber 13 , and the culture solution drain tube 12 is connected to the bottom end of the central guide tube 21 .

In practical applications, the shapes of the first fixing frame 22 and the second fixing frame 23 are not too restrictive, preferably semi-cylindrical; the shapes of the first culture plate 24 and the second culture plate 25 can also not be too restrictive Generally speaking, the second culture plate 25 corresponding to the semi-circular first culture plate 24 is more preferred, but other shapes such as semi-elliptical ring, semi-hollow rectangular column, etc. are selected.

In the application of cell expansion in vitro, the cell carrier needs to be taken out for the next step after the cells are congested. The existing multi-layer plate bioreactor is installed and disassembled layer by layer according to the order of the number of plate layers, and the disassembly efficiency is low, thereby increasing the risk of cell contamination.

Therefore, the first fixed frame 22 and the second fixed frame 23 that can be opened and closed in this embodiment are fixed with a plurality of first culture plates 24 and second culture plates 25 in a detachable manner, which can realize the first fixed The frame 22 and the second fixed frame 23 improve the pick-and-place speed of the first culture plate 24 and the second culture plate 25, which is very important for the continued maintenance of cell viability and cell activity.

In this embodiment, one end of the first fixing frame 22 and one end of the second fixing frame 23 are hingedly connected. In practical applications, the first fixing frame 22 and the second fixing frame 23 that are matched in an openable and closable manner can also be connected in other ways, such as clamping at both ends.

Preferably, the first fixing frame 22 is provided with a first engaging portion 29 for engaging the first culture plate 24, and the second fixing frame 23 is provided with a locking portion for engaging the second culture plate 25. The second engaging portion 20 ; the central guide tube 21 is provided with a third engaging portion 200 for engaging with the first concave portion 26 and the second concave portion 27 . The cooperation of the first engaging part 29 and the third engaging part 200 realizes the stable fixing of the first culture plate 24 in use; the cooperation of the second engaging part 20 and the third engaging part 200 realizes the second culture plate 25 in the Stable fixation while in use.

More preferably, the other end of the first fixing frame 22 and the other end of the second fixing frame 23 are fixedly connected by a snap-fit rack 201 .

In this embodiment, an observation mechanism 3 is also included. The observation mechanism 3 is arranged on the perfusion cavity 1 and is located directly above the culture mechanism 2. The observation mechanism 3 includes a watertight observation mechanism arranged on the perfusion cavity 1. The window 31 and the imaging device 32 detachably arranged above the watertight observation window 31 ; the imaging device 32 is provided with an auxiliary light source 33 . More specifically, the imaging device 32 is composed of an industrial camera, a height adjuster, a magnification adjuster, and an objective lens. The auxiliary light source 33 is generally selected as an LED light source, and the auxiliary light source 33 is arranged below the objective lens. The objective lens is an ordinary optical microscope objective lens or a phase contrast microscope objective lens with a magnification of 0.5-5.0x. The industrial camera is a 10 million high-resolution camera, and the image is transmitted through a common video interface such as USB or VGA.

The cooperation of the watertight observation window 31 and the imaging device 32 in this embodiment facilitates disassembly of the imaging device 32 without causing bacterial contamination. The watertight observation window 31 is composed of the upper cover of the observation window, the connector, the bulkhead, the transparent glass sheet and the lower cover of the observation window. Except for the glass sheet, the above-mentioned parts are made of 316L stainless steel, and each part is watertight through threads and silicone O-rings. Connect and fix to the upper part of the perfusion chamber 1 in the same way. The watertight observation window 31 has an overall height of 112 mm, an inner diameter of 46 mm, and an outer diameter of 50 mm, and its lower part is directly facing the cultivation mechanism 2 with a multi-layer plate structure.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (10)

1. a kind of multi-layer planar filling type bioreactor, including perfusion cavity and to be arranged on filling cavity in vivo by multi-layer planar The culture mechanism of composition, the perfusion cavity are equipped with culture solution inlet tube and culture solution drain pipe, which is characterized in that institute The perfusion cavity stated is divided into culture chamber and cushion chamber, and the culture mechanism is arranged in culture chamber, the culture solution into The outlet of liquid pipe is extended to below the middle part of cushion chamber, and the top of the cushion chamber is connected with culture chamber.

2. multi-layer planar filling type bioreactor according to claim 1, which is characterized in that on the perfusion cavity Equipped with the conduit with atmosphere.

3. multi-layer planar filling type bioreactor according to claim 1, which is characterized in that the culture mechanism bag The first fixed frame and the second fixed frame for including center diversion pipe and being coordinated in a manner of openable and closable, on first fixed frame Multiple first culture plates are from top to bottom detachably secured, from top to bottom with removable on second fixed frame The mode unloaded is fixed with multiple the second culture plates corresponding with the first culture plate, and first culture plate is equipped with the One recess portion, second culture plate are equipped with the second recess portion；

When the first fixed frame and the second fixed frame are closed, the first recess portion of the first culture plate and the second of the second culture plate Recess portion forms the ring with the cooperation of center diversion pipe；

The center diversion pipe is equipped with the through hole connected with culture chamber, and the culture solution drain pipe is connected to center water conservancy diversion The bottom of pipe.

4. multi-layer planar filling type bioreactor according to claim 3, which is characterized in that first fixed frame One end and the second fixed frame one end articulated connection.

5. multi-layer planar filling type bioreactor according to claim 3, which is characterized in that first fixed frame It is equipped with to engage the first holding section of the first culture plate, second fixed frame is equipped with to engage the second culture Second holding section of tablet；The center diversion pipe is equipped with for the 3rd card with the first recess portion and the clamping engagement of the second recess portion Conjunction portion.

6. according to any multi-layer planar filling type bioreactors of claim 3-5, which is characterized in that described first Culture plate and the second culture plate are semicircle.

7. multi-layer planar filling type bioreactor according to claim 4, which is characterized in that first fixed frame The other end and the other end of the second fixed frame be fixedly connected by being clamped rack.

8. multi-layer planar filling type bioreactor according to claim 1, which is characterized in that sampling mechanism is further included, The sampling mechanism is arranged on perfusion cavity and directly over culture mechanism.

9. multi-layer planar filling type bioreactor according to claim 8, which is characterized in that the sampling mechanism bag Include the watertight observation window being arranged on perfusion cavity and the imaging device being removeably positioned above watertight observation window； The imaging device is equipped with secondary light source.

10. multi-layer planar filling type bioreactor according to claim 9, which is characterized in that the culture mechanism Multi-layer planar be high light-transmitting materials multi-layer planar.