WO2017152343A1

WO2017152343A1 - Recirculating perfusion bioreactor device that can realize three-dimensional scaffold recirculating perfusion

Info

Publication number: WO2017152343A1
Application number: PCT/CN2016/075779
Authority: WO
Inventors: 李君�; 李兰娟; 周倩
Original assignee: 浙江大学
Priority date: 2016-03-07
Filing date: 2016-03-07
Publication date: 2017-09-14

Abstract

Provided is a recirculating perfusion bioreactor device, comprising a bioreactor and a perfusion device, wherein the perfusion device comprises a peristaltic pump and a pipe system arranged on the peristaltic pump, the bioreactor comprises a sterile culture chamber and an airtight matching cap, and a three-dimensional scaffold can be connected to the interior of the bioreactor.

Description

Circulating perfusion bioreactor device capable of realizing three-dimensional stent circulation perfusion

Technical field

The invention belongs to the fields of clinical medicine, tissue engineering and regenerative medicine, in particular, is a new device capable of realizing continuous supply of oxygen and nutrients of seed cells in tissue engineering three-dimensional scaffold.

Background technique

Tissue engineering is a combination of engineering and life science principles and methods. It conducts the most basic research on the relationship between mammalian tissue structure and function in both normal and pathological states, and develops biologically useful alternatives for repair. A discipline that maintains or improves the function of damaged tissues or organs. The basic principle is: the normal tissue cells are adsorbed on the biocompatible biomaterial to form a complex. After a period of cultivation, the cells are expanded and the biomaterial is gradually degraded and absorbed, thereby forming a specific morphology, structure and function. Corresponding tissues and organs achieve the purpose of promoting tissue regeneration, repairing wounds and reconstructing functions. At the heart of tissue engineering is the creation of a three-dimensional complex of cells and biological materials. As a carrier of tissue engineering, scaffolding is a key part of tissue engineering research. It not only provides a three-dimensional environment for cell growth and a place for metabolism, but also determines the shape and size of new tissue and organs. Among them, the decellularized scaffold has become a research hotspot in the field of tissue engineering because it retains the intact vascular network structure and extracellular matrix components. At present, with the development of tissue engineering technology and materials science, some simple tissue organs, such as skin, blood vessels, bones, cartilage, etc., can be artificially constructed, bringing dawn to the treatment of organ transplant patients. However, organ organizations with relatively complex structures and functions still face enormous challenges. Among them, the continuous and effective supply of cellular oxygen and nutrients in large three-dimensional scaffolds is a major problem. How to achieve long-term culture and functional maintenance of cells in three-dimensional scaffolds is our key consideration. The emergence of tissue engineering bioreactors provides technical support for the construction of tissue engineering organs.

In the development of tissue engineering bioreactors, flat bioreactors, rotary bottle bioreactors, hollow fiber bioreactors, perfusion bed scaffold bioreactors, seed cell packs have emerged. Wrap/suspension bioreactors, etc. However, the above reactor cannot solve the problem of supply of oxygen and nutrients in the cells of the large three-dimensional tissue module, and its applicability is limited, mainly for cells, microcapsules, and tiny tissue modules. To this end, it is necessary to consider when constructing a bioreactor suitable for large three-dimensional scaffolds: 1) perfusate can affect seed cells deep inside the three-dimensional scaffold; 2) provide continuous perfusion to simulate physiological conditions in vivo; 3) provide Simulate the biological and mechanical environment and sufficient nutrient exchange in the physiological environment of the body. The continuous circulation perfusion of the perfusate in the stent is achieved by adjusting the perfusion rate and the support of the pressure negative feedback regulation system.

The invention is based on the improvement of the existing circulating perfusion bioreactor, and provides a simple circulating perfusion device for culturing tissue engineering organs in vitro, and provides technical support for the research of stem cell differentiation and cell growth under three-dimensional conditions.

Summary of the invention

The invention aims at the problem that the cells in the tissue engineering three-dimensional scaffold can not survive for a long time due to insufficient supply of oxygen and nutrients, and the activity rate is low, and a cyclic perfusion bioreactor device is disclosed to provide continuous cells for the three-dimensional scaffold. The supply of nutrients provides technical support for the study of stem cell differentiation and cell growth under three-dimensional conditions, overcomes the shortcomings of existing static culture, and constructs functional tissue engineering organs. Thereby, the long-term culture and function maintenance of the cells in the three-dimensional scaffold are realized, the tissue engineering organ is constructed, and the long-term culture of the recellularized scaffold in vitro is realized while ensuring the cell activity.

The present invention is achieved by the following technical solutions:

The invention discloses a circulating perfusion bioreactor device. The device comprises a bioreactor and a perfusion device. The perfusion device comprises a peristaltic pump and a pipeline system arranged on the peristaltic pump. The bioreactor comprises a sterile culture chamber and a closed anastomosis cover. The bioreactor can be connected with a three-dimensional scaffold to meet the required culture space of the three-dimensional scaffold, avoiding a large waste of the culture liquid, facilitating the operation of the three-dimensional scaffold in the sterile chamber, storing the culture medium in the sterile culture chamber, and placing the three-dimensional scaffold, and peristaltic The pump can adjust the control by setting the perfusion mode, perfusion flow rate, irrigation flow rate, etc., to adapt to different research needs and organizational construction needs.

As a further improvement, the bioreactor of the present invention has a liquid input end on the reactor, and a sealed inlet cap is provided with a liquid input end of the reactor and a gas inlet, and the liquid output end of the reactor is placed. Below the liquid level in a sterile culture chamber.

As a further improvement, the pipeline system of the present invention is a silica gel tube-like structure comprising a silica gel inflow section and a silica gel outflow section, and the inlet end of the piping system on the silica inflow section is connected to the liquid output end of the reactor, and the silica gel is discharged from the section. The outlet end of the piping system is connected to the liquid input end of the reactor. Thereby forming a closed circulating perfusion bioreactor device;

As a further improvement, a gas filter is connected to the gas inlet of the present invention. The gas filter can ensure the sterilizing of oxygen, carbon dioxide and the like into the sterile culture chamber outside the reactor to ensure the sterility of the sterile culture chamber, and at the same time satisfy the sufficient gas supply of the culture liquid.

As a further improvement, the liquid input end of the reactor and the liquid output end of the reactor of the present invention are detachable and independent tubular structures. The design avoids the over-tightness of the closed anastomosis cap and the good compatibility with the bioreactor, which ensures the tightness of the entire reactor system.

As a further improvement, the closed anastomosis cover of the present invention and the sterile culture chamber body portion are provided as a matte. The sealing of the reactor unit is guaranteed.

As a further improvement, the liquid input end of the reactor of the present invention is fine at both ends, and is respectively connected with a silicone tube and a trocar, and the intermediate diameter is increased to exhibit a drip-like structure, and the liquid input end of the reactor is It is at an angle of 70-80° to the side wall of the reactor. Instead of entering vertically, on the one hand, the design allows the gas generated in the silicone pipe system to be above the liquid level in the input pipe, thereby effectively preventing air bubbles from entering the bracket in the silicone pipe system. On the other hand, the inflow port of the three-dimensional support can be connected to the liquid input end on the side wall without causing the tear deformation of the support.

As a further improvement, the bioreactor of the present invention is made of a transparent material or a disposable sterile material. The transparent material is heat-resistant and high-temperature resistant, can withstand high-pressure steam sterilization of 120-200 degrees, realizes repeated use of the reactor device, and the transparent design of the container can be more conveniently observed. The disposable sterile material is a transparent, non-toxic, stable and stable disposable material, which is convenient, high-efficiency, avoids cross-contamination, does not release toxic substances in the cell culture environment, affects cell growth, and the transparent design of the container It is also easy to observe.

As a further improvement, the transparent material of the present invention is transparent borosilicate glass or quartz glass.

As a further improvement, the piping system of the present invention is a high pressure steam sterilized silicone tube that can withstand 120-200 degrees. The silicone tube is resistant to high temperatures and stable in performance.

The present invention has several advantages over existing liver tissue engineering bioreactors:

First, the bioreactor sterile culture chamber culture liquid enters the closed pipeline system through the liquid output end of the reactor, and under the control of the peristaltic pump, flows into the three-dimensional stent through the liquid input end of the reactor, thereby realizing the culture liquid in Complete circulation in the entire three-dimensional scaffold provides effective nutrient supply, gas exchange, enhanced cell survival rate in the scaffold, and recellularization efficiency of the three-dimensional scaffold. In addition, the pulsation stimulation of the peristaltic pump simulates blood flow in the body.

Secondly, the gas inlet is arranged on the reactor, and a gas filter is arranged thereon to ensure that the oxygen, carbon dioxide and the like gas entering the sterile culture chamber outside the reactor are aseptic, so as to satisfy the sufficient gas supply of the culture liquid.

Thirdly, the liquid input end of the reactor draws on the design principle of the disposable infusion tube "Mofite dropper", and is designed to be small at both ends, which are respectively connected with the silicone tube and the trocar, and the intermediate diameter is increased to present a drip-like structure. . The liquid input end is at an angle of 70-80° to the side wall of the reactor instead of entering vertically. On the one hand, the gas generated in the silica gel pipe system is located above the liquid level in the input end tube, thereby effectively avoiding the silicone pipe. Air bubbles in the system enter the bracket. On the other hand, the design allows the three-dimensional support to be connected to the liquid input end of the side wall without causing the tensile deformation of the support.

Fourth, the whole perfusion system is simple and easy to operate. The culture fluid perfusion in the stent can be controlled by adjusting and setting the perfusion mode, perfusion flow rate, perfusion pressure on the peristaltic pump.

DRAWINGS

Figure 1 is a schematic diagram showing the principle structure of an extracorporeal circulation perfusion bioreactor device;

In the figure, 1 is a bioreactor, 2 is a perfusion device, 3 is a sterile culture chamber, 4 is a piping system, 5 is a liquid input end of the reactor, 6 is a liquid output end of the reactor, 7 is a gas inlet, 8 At the inlet end of the piping system, 9 is the outlet end of the piping system, 10 is a gas filter, and 11 is a closed sealing cover.

detailed description

1 is a schematic structural diagram of a cardiopulmonary perfusion bioreactor device; the present invention discloses a circulating perfusion bioreactor device comprising a bioreactor 1 and a perfusion device 2, the perfusion device 2 comprising a peristaltic pump and being placed in a peristaltic The piping system 4 on the pump, the bioreactor 1 comprises a sterile culture chamber 3 and a closed anastomosis cover 11. The liquid inlet end 5 of the reactor is opened on the side wall of the bioreactor 1, and the liquid inlet end 5 and the gas inlet port 7 of the reactor are opened on the closed anastomosis cover 11. The pipe system 4 is a silicone tube-like structure. Including the silica inflow section and the silica outflow section, the inlet end 8 of the piping system on the silica inflow section is connected to the liquid output end 6 on the reactor, and the outlet end 9 of the piping system on the silica outflow section is connected to the liquid input end 5 on the reactor, the gas A gas filter 10 is connected to the inlet 7, and the liquid input end 5 on the reactor and the liquid output end 6 on the reactor are detachable and independent tubular-like structures, and the closed anastomosis cover 11 and the sterile culture chamber 3 are arranged at an anastomosis. For the sanding, the liquid input end 5 of the reactor is fine at both ends, respectively connected to the silicone tube and the trocar, and the intermediate diameter is increased to exhibit a drip-like structure, and the liquid input end 5 and the reactor side wall of the reactor are arranged. At an angle of 70-80°, the bioreactor 1 is made of transparent material or disposable sterile material. The transparent material is transparent borosilicate glass or quartz glass, and the pipe system 4 can withstand 120-200 degrees. High-pressure steam-sterilized silicone tube, disposable sterile material is transparent, non-toxic, stable in performance, and does not release toxic substances in cell culture environment.

The circulating perfusion bioreactor 1 system disclosed in the present invention simulates blood flow in the body and provides nutrients necessary for long-term culture of cells in the stent. Both the circulating perfusion device 2 and the bioreactor 1 are placed in a carbon dioxide incubator. The liquid output end 6 of the reactor penetrates below the liquid level, and the liquid in the sterile culture chamber 3 is pumped out by the peristaltic pump outside the reactor, through the closed silicone pipe system 4 and the liquid input connected thereto. It flows into the three-dimensional scaffold and then flows out into the sterile culture chamber 3 in the reactor through the outflow port of the three-dimensional scaffold, thereby realizing continuous circulation perfusion culture of the three-dimensional scaffold. The three-dimensional scaffold is a biocompatible material having a liquid inflow port and an outflow port, and the intact vasculature is retained, and the trocar is ligated with the liquid inflow port of the scaffold to form a liquid inlet tube of the three-dimensional scaffold, The liquid inlet pipe is connected to the liquid input end 5 of the reactor, and the liquid outflow port of the three-dimensional support is directly opened to the sterile culture chamber 3.

The present invention further clarifies the technical scheme of the present invention by taking a whole liver decellularized scaffold as an example, and performing specific embodiments from two stages of whole liver decellularized scaffold recellularization and in vitro culture:

The whole liver decellularization scaffold recellularization stage, that is, the cell planting stage. The whole liver decellularized scaffold obtained by the detergent rinsing is placed in a sterile dish, and the seed cells are suspended in 5 ml of the culture solution, and slowly injected into the stent through the portal vein, and the culture is allowed to stand in the incubator for a period of time. Tissue perfusion culture is performed.

In the in vitro culture stage, the circulating perfusion device 2 and the bioreactor 1 are both placed in a carbon dioxide incubator. The whole liver acellular scaffold is cultured in the sterile culture chamber 3 of the bioreactor 1, and the culture medium in the sterile culture chamber 3 enters the closed conduit system 4 through the liquid output end 6 of the reactor deep into the liquid level, under the control of the peristaltic pump The culture solution is subjected to a three-dimensional stent of the whole liver through the liquid input end 5 of the reactor. The culture medium enters the sterile culture chamber 3 after circulating in the whole liver three-dimensional stent, and repeats the above-mentioned circulation process, thereby realizing the completion of the culture solution in the three-dimensional stent. Full cycle.

Example 1: Seed cells were cultured in whole liver decellularized scaffolds

Under sterile conditions, the portal vein was seen in the middle of the abdomen of the rat, and the trocar was inserted through the portal vein for subsequent continuous perfusion of the detergent to obtain a rat whole liver acellular stent, which retained the intact vessel. Structure, as well as extracellular matrix components. The seed cells were suspended in 5 ml of the culture medium, and were injected into the stent through a trocar connected with a portal vein ligation. The culture was allowed to stand in the culture dish for a period of time, and then the whole liver was perfused and cultured. The culture solution is added to the sterile culture chamber 3 of the bioreactor 1, and the trocar connected to the portal vein on the whole liver stent is connected to the liquid input end 5 of the reactor, wherein the liquid input end 5 of the reactor and the reactor side wall are At an angle of 75°, the liquid outflow port of the whole liver stent is directly opened in the sterile culture chamber 3. The liquid output end 6 of the reactor penetrates into the liquid level in the sterile culture chamber 3, and the inlet end 8 of the piping system on the silica inflow section is connected to the liquid output end 6 of the reactor, and the outlet end of the piping system on the silica gel outlet section is reacted. The liquid inlets 5 are connected to form a closed circulating perfusion bioreactor unit. The gas filter 10 on the closed anastomosis cover 11 ensures that the gas entering the bioreactor 1 is sterile. Under the control of the peristaltic pump, the circulating perfusion of the culture fluid in the whole liver stent is realized, and the supply of oxygen and nutrients of the seed cells in the decellularized stent is ensured. The entire set of circulating perfusion bioreactor 1 is placed in a carbon dioxide incubator.

Example 2: Effect of different perfusion rates on seed cells in whole liver stents

Under sterile conditions, the portal vein was seen in the middle of the abdomen of the rat, and the trocar was inserted through the portal vein for subsequent continuous perfusion of the detergent to obtain a rat whole liver acellular stent, which retained the intact vessel. Structure, as well as extracellular matrix components. The seed cells were suspended in 5 ml of the culture medium, and were injected into the stent through a trocar connected with a portal vein ligation. The culture was allowed to stand in the culture dish for a period of time, and then the whole liver was perfused and cultured. The culture solution is added to the sterile culture chamber 3 of the bioreactor 1, and the trocar connected to the portal vein on the whole liver stent is connected to the liquid input end 5 of the reactor, wherein the liquid input end 5 of the reactor and the reactor side wall are At an angle of 75°, the liquid outflow port of the whole liver stent is directly opened in the sterile culture chamber 3. The liquid output end 6 of the reactor penetrates into the liquid level in the sterile culture chamber 3, and the inlet end 8 of the piping system on the silica inflow section is connected to the liquid output end 6 of the reactor, and the outlet end of the piping system on the silica gel outlet section is reacted. The liquid inlets 5 are connected to form a closed circulating perfusion bioreactor unit. The gas filter 10 on the closed anastomosis cover 11 ensures that the gas entering the bioreactor 1 is sterile. Continuous perfusion culture at different perfusion rates was performed by adjusting the perfusion rate of the peristaltic pump. After one week of culture, observe and compare different perfusions The effect of speed on seed cells in acellular scaffolds.

Example 3: Differentiation and culture of stem cells in acellular scaffold

Under sterile conditions, the portal vein was seen in the middle of the abdomen of the rat, and the trocar was inserted through the portal vein for subsequent continuous perfusion of the detergent to obtain a rat whole liver acellular stent, which retained the intact vessel. Structure, as well as extracellular matrix components. Bone marrow mesenchymal stem cells (BMSCs) were suspended in 5 ml of DMEM containing 10% FBS, and the whole liver decellularized scaffold was recellularized by a trocar connected with a portal vein ligation. The whole cultured circulatory perfusion culture was carried out after standing culture for a period of time in the culture dish. The culture solution is added to the sterile culture chamber 3 of the bioreactor 1, and the trocar connected to the portal vein on the whole liver stent is connected to the liquid input end 5 of the reactor, wherein the liquid input end 5 of the reactor and the reactor side wall are At an angle of 75°, the liquid outflow port of the whole liver stent is directly opened in the sterile culture chamber 3. The liquid output end 6 of the reactor penetrates into the liquid level in the sterile culture chamber 3, and the inlet end 8 of the piping system on the silica inflow section is connected to the liquid output end 6 of the reactor, and the outlet end of the piping system on the silica gel outlet section is reacted. The liquid inlets 5 are connected to form a closed circulating perfusion bioreactor unit. The gas filter 10 on the closed anastomosis cover 11 ensures that the gas entering the bioreactor 1 is sterile. Under the control of the peristaltic pump, the circulating perfusion of the culture fluid in the whole liver stent is realized, and the supply of oxygen and nutrients of the seed cells in the decellularized stent is ensured. Several days after the expansion of BMSCs, the cells were cultured for 2 days in serum-free IMDM medium, followed by hepatic differentiation culture of whole liver decellularized stem cells, collected at 3 days, 1, 2, and 3 weeks after circulating culture. Tissue and culture supernatant were analyzed to identify the differentiation effect of stem cells in decellularized scaffolds, and the effect of decellularized scaffolds on stem cell differentiation was evaluated.

Continuous circulation perfusion plays a key role in the supply of nutrients and nutrients in seed cells and is a key factor in regulating cell growth and maintaining functional activity. The bioreactor of the invention is simple and convenient to operate, and can provide the cardiomyocytes in the whole liver decellularization scaffold with a continuous simulated physiological condition of the extracorporeal circulation, providing sufficient gas and nutrients for the seed cell growth, and recellularizing the decellularized scaffold. Provide technical support to provide the means to explore the best tissue culture techniques.

The above are only the preferred embodiments of the present invention, and the present invention is not limited to the above embodiments, and other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention. It is considered to be included in the scope of protection of the present invention.

Claims

A circulating perfusion bioreactor device, characterized in that the device comprises a bioreactor (1) and a perfusion device (2), the perfusion device (2) comprising a peristaltic pump and a pipe arranged on the peristaltic pump System (4), said bioreactor (1) comprises a sterile culture chamber (3) and a closed anastomosis cover (11).
The circulatory perfusion bioreactor device according to claim 1, characterized in that the side wall of the bioreactor (1) is provided with a liquid input end (5) on the reactor, and the closed sealing cap ( 11) The upper liquid inlet (5) and the gas inlet (7) are provided on the reactor.
The circulatory perfusion bioreactor device according to claim 2, wherein the pipe system (4) is a silicone tube-like structure comprising a silica gel inflow section and a silica gel outflow section, and the silica gel inflow section is a pipe. The system inlet end (8) is connected to the liquid output end (6) of the reactor, and the outlet end (9) of the piping outlet on the silica gel outlet section is connected to the liquid input end (5) of the reactor.
A circulating perfusion bioreactor apparatus according to claim 2, characterized in that a gas filter (10) is connected to the gas inlet (7).
The circulating perfusion bioreactor device according to claim 1 or 2 or 3 or 4, characterized in that the liquid input end (5) on the reactor and the liquid output end (6) on the reactor are detachably replaceable Independent tubular structure.
The circulatory perfusion bioreactor device according to claim 1 or 2 or 3 or 4, characterized in that the closed portion of the closed anastomosis cap (11) and the sterile culture chamber (3) is set to a matte.
The circulatory perfusion bioreactor device according to claim 1 or 2, wherein the liquid input end (5) of the reactor is thin at both ends, and is respectively connected with a silicone tube and a trocar, and the intermediate diameter is increased. The drip-like structure is presented, and the liquid input end (5) on the reactor is at an angle of 10-20° to the side wall of the reactor.
A circulating perfusion bioreactor device according to claim 1 or 2 or 3 or 4, characterized in that the bioreactor (1) is made of a transparent material or a disposable sterile material.
The circulatory perfusion bioreactor apparatus according to claim 1 or 2 or 3 or 4, wherein the transparent material is transparent borosilicate glass or quartz glass.
A circulatory perfusion bioreactor apparatus according to claim 1 or 2 or 3 or 4, wherein said piping system (4) is a high pressure steam sterilized silicone tube that can withstand 120-200 degrees.