CN104911104B - In-vitro cell and organ 3-D culture device and artificial organ culture method - Google Patents

Abstract

The invention discloses a 3-D culture device in vitro for cells and organs and a method for cultivating artificial organs, and relates to the technical field of microbiology. The liquid circulation system and the gas exchange system also include a multi-degree-of-freedom motion mechanism. The method comprises using a 3-D culture device in vitro of cells and organs to simulate the dynamic environment of growth and development of human organs to cultivate artificial organs. The present invention more realistically simulates the growth microenvironment of human cells or organs developing fetus in the mother by setting a 3-D motion shaker, and improves the growth and differentiation efficiency of various stem cells into functional cells in 3-D culture in vitro , to provide a simple, economical, mild, three-dimensional, and nutritionally comprehensive growth microenvironment for 3-D culture of cells in vitro. Experiments show that the method of using this device for in vitro cultivation of human organs is feasible and can be applied clinically.

Description

Cell and organ in vitro 3-D culture device and method for artificial organ culture

technical field

The invention belongs to the technical field of microbiology, and relates to an in vitro 3-D culture device for cells and organs and a cultivation method for artificial organs.

Background technique

In order to avoid the drawbacks of cells or tissues gradually losing many physiological characteristics of the original tissue during two-dimensional (2-D) growth, in vitro three-dimensional (3-D) culture was invented. Currently used in vitro 3-D culture devices mainly use bioreactor (Bioreactor, BR), which refers to a device system or engineering equipment that uses the biological functions of enzymes or organisms (such as microorganisms) to provide a biologically active environment in vitro. According to the different mixing methods of cultured cells, culture carriers, and culture medium, BR is mainly divided into stirring type, gas type, hollow fiber type, and rotary type (space BR). According to different structures, BR is divided into the box structure of rotating technology combined with ordinary 37°C 5% CO2 cell incubator; the box structure of circulating culture device and ordinary 37°C 5% CO2 cell incubator, circulating culture device and ordinary 37°C 5 The %CO2 cell incubator is an independent device and a closed device for circulating culture combined with gas exchange. In addition, the supporting technologies for BR include perfusion culture, microcarrier, porous microsphere, etc.

However, from the perspective of economic benefits, these BRs generally have high cost and complicated equipment. For example, from the functional point of view, the shear force generated by the stirring blades of the stirring BR can cause damage to the cells; the cultivation environment of the hollow fiber BR is not uniform, the cultivation process is not easy to scale up, and it is difficult to sterilize and reuse; from the structural point of view, the structural one The device is expensive and bulky, which undoubtedly increases the difficulty of purchasing and using the instrument; the structure 2 device is expensive and bulky, and the 3-D culture tube in the device is fixed in the incubator and cannot be rotated, so it cannot be quickly It is good to realize the three-dimensional culture function of cells or tissues growing in a natural suspension movement similar to the human body development process, resulting in the loss of function or dysplasia of artificial organs; although the swing or rotation system in the three-structure device can make cells 3-D culture in a dynamic process , but because the 3-D culture tube is placed outside the incubator, the probability of contamination is greatly increased, and the circulation culture combined with the gas exchange closed device requires a high-level surrounding culture atmosphere with high cost.

In order to overcome the above problems, there is an urgent need to improve the structure of the existing culture device. The improved device can not only simulate the compound movement of the fetus during the development of amniotic fluid in the placenta of the mother, but also carry out the cyclic exchange of metabolites, which can fully meet the needs of high-density cells. Value-added needs, but also to ensure efficient secretion of cells, to provide a simple, economical, gentle, three-dimensional, nutritionally comprehensive 3-D growth microenvironment for in vitro cultivation of cells or organs, and to promote cell regeneration and the generation of artificial organs.

Contents of the invention

In view of this, one of the purposes of the present invention is to provide an in vitro 3-D culture device for cells and organs to solve the problem that existing technologies cannot truly simulate the growth microenvironment of human organs, resulting in dysplasia, loss of function, and low cultivation efficiency of artificial organs. The problem. The second object of the present invention is to provide a method for artificially cultivating organs using the above-mentioned in vitro 3-D culture device for cells and organs.

One of purpose of the present invention is achieved through the following technical solutions:

The 3-D culture device for cells and organs in vitro of the present invention comprises a culture room, a 3-D motion shaking box capable of compound motion, and a conditional culture solution circulation system and a gas exchange system respectively communicated with the culture room. It is detachably suspended in the 3-D motion shaker through elastic components, and also includes a multi-degree-of-freedom motion mechanism arranged on one side or both sides of the 3-D motion shaker.

Further, the culture environment of the culture room: the temperature is 37°C; the humidity is 95-98%; the air contains 5% CO ₂ by volume; the pH value of the acid-base is 7.2-7.4.

Further, the multi-degree-of-freedom motion mechanism includes a longitudinal rotation mechanism, a horizontal rotation mechanism, and a multifunctional connection structure arranged between the two, and the conditional culture fluid circulation system and the gas exchange system are connected to the outside world through the multifunctional connection structure. Culture medium and gas exchange.

Further, the longitudinal rotation mechanism includes a longitudinal rotation shaft and a speed-regulating motor 1, one end of the longitudinal rotation shaft is fixed with the 3-D motion shaker, and the other end is fixed with the main shaft of the speed-regulation motor 1.

Further, the horizontal rotation mechanism includes a horizontal rotation frame, a horizontal rotation shaft and a speed-regulating motor II, one end of the horizontal rotation frame is rotatably connected to one end of the longitudinal rotation shaft, and the other end is rotatable connection to one end of the horizontal rotation shaft. The other end of the horizontal rotating shaft is fixed with the main shaft of the speed-regulating motor II.

Further, the joints between the horizontal rotating frame and the longitudinal and horizontal rotating shafts are provided with multifunctional connecting structures.

Further, the multi-degree-of-freedom motion mechanism is arranged on both sides of the 3-D motion shaker, and the multi-degree-of-freedom motion mechanism also includes a longitudinal driven shaft fixedly arranged on the other side of the 3-D motion shaker, the A multifunctional connecting structure is arranged between the longitudinal driven shaft and the horizontal rotating frame.

Further, the multifunctional connection structure includes a movable sleeve that is rotatable and hermetically fitted on the outer surface of the longitudinal or horizontal rotating shaft, a sealing chamber arranged between the movable sleeve and the longitudinal or horizontal rotating shaft, and a sealing chamber arranged between the movable sleeve and the longitudinal or horizontal rotating shaft. The rolling bearings between the horizontal rotation shafts, and the conditional culture solution circulation channel and gas exchange channel arranged in the horizontal rotation shaft, horizontal rotation frame, longitudinal rotation shaft and longitudinal driven shaft, the sealed cavity passes through the conditional culture solution circulation channel and The gas exchange channels communicate with the culture chambers respectively.

Further, a plurality of baffles made of elastic material are evenly spaced in the cultivation chamber, and elastic buffer films with arc tops are provided on the heads of the baffles.

Further, the elastic component is a spring or rubber or other elastic components.

Further, the culture chamber includes a chamber body and a chamber cover that can seal and fix the chamber body, the chamber cover is provided with a locking structure that fits with the chamber body, and the chamber body is provided with a matching structure. Snap fit construction.

Further, it also includes a PLC control system and an information display system for fully automatic control of the cultivation device.

Two of the purpose of the present invention is achieved through the following technical solutions:

Using the method for cultivating artificial organs with the above-mentioned in vitro cell and organ 3-D culture device, the artificial organ is cultivated by using the cell and organ in vitro 3-D culture device to simulate the dynamic environment of human organ growth and development.

Further, the dynamic environment for the growth and development of human organs includes: elastically suspending the natural decellularized whole organ scaffold of the seed cells in a 3-D culture device, and stage-by-stage at 37°C and 5% CO ₂ Cultivation, the first stage is 1-7 days, the second stage is 8-14 days, and the third stage is 15-21 days. 24h dynamic culture is adopted. During the day, it first rotates 360° to the left horizontally, and then rotates 360° to the right horizontally. The composite 3-D rotation combining vertical rotation and horizontal rotation, the rotation speed is 20-40rpm/min during the day, and the rotation speed is less than 20rpm/min at night, the medium is changed every three days, and different mediums are replaced at each stage.

Beneficial effects of the present invention:

1. The 3-D culture device for cells and organs in vitro of the present invention simulates the compound movement of human body development of the fetus in the process of amniotic fluid development of the mother's placenta by setting a 3-D motion shaker, and is elastically arranged in the shaker by the culture chamber Simulate the simple harmonic motion of the culture during the suspension process in the human body, more realistically simulate the growth microenvironment of human cells or organs during the development process, improve the differentiation of various seed cells into mature cell density and culture efficiency, and provide cells Or the in vitro 3-D culture of organs provides a simple, economical, mild, three-dimensional, nutritionally comprehensive growth microenvironment to accelerate the generation of in vitro artificial organs. Compared with the existing technology, this device has obvious cost advantages and promotion value.

2. Through the multifunctional connection structure, the purpose of relative rotation between the medium input system and the rotating shaft can be realized, and since each medium channel is set on the rotating shaft, the 3-D motion shaker and the culture room can realize the medium (culture solution) required for the growth environment. and 37°C 5% CO ₂ gas) are communicated and exchanged with the cultivation chamber, which solves the problem of input pipeline winding during multi-degree-of-freedom rotation.

3. The method for cultivating artificial organs of the present invention can realize in vitro cultivation and reconstruction of various human organs because the device truly simulates the growth and development microenvironment of human cells or organs. It takes only 3 weeks to culture the liver-like tissue formed by the method of the present invention, which is 1 week less than the liver-like tissue (requiring 4 weeks) induced by the BR method in vitro to form the liver-like tissue formed by DLS mesenchymal stem cells (MSCs) in vitro. Time, experiments show that the artificial liver prepared by the present invention has liver structure and function, and can be used as a liver transplantation donor. It is further confirmed that the method of using this device for in vitro cultivation of human organs is feasible and can be applied clinically, and has a wide range of applications. application prospects and high social value.

Other advantages, objects and features of the present invention will be set forth in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the investigation and research below, or can be obtained from It is taught in the practice of the present invention. The objects and other advantages of the invention may be realized and attained by the following specification.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with the accompanying drawings, wherein:

Accompanying drawing 1 is the structural representation of the in vitro cell and organ 3-D culture device of the present invention;

Accompanying drawing 2 is the enlargement figure of cultivation room of the present invention;

Accompanying drawing 3 is the enlarged view of multifunctional connecting structure I of the present invention;

Accompanying drawing 4 is the enlarged view of multifunctional connecting structure II of the present invention;

Accompanying drawing 5 is the enlarged view of the multifunctional connecting structure III of the present invention.

Accompanying drawing 6 is the artificial liver cultivated with this device. A: Hepatic lobe-like structures formed on the 9th day of in vitro 3-D culture; B: Whole liver-like structures were formed on the 21st day of in vitro 3-D culture; C(a-b) tissue sections showed hepatic lobule-like structures similar to normal liver; C(c) secretes liver function proteins.

Accompanying drawing 7 is the schematic diagram of mammalian liver development and maturation. A is mouse; B is human.

Reference signs: 1-cultivation room; 2-3-D motion shaking box; 3-longitudinal rotation shaft; 4-speed regulating motor I; 5-horizontal rotating frame; 6-horizontal rotating shaft; 7-speed regulating motor II; 8-longitudinal driven shaft; 9-movable sleeve; 10-rolling bearing; 11-culture solution circulation channel; 12-gas exchange channel; 13-seal chamber I; 14-seal chamber II; 15-seal chamber III; 16-seal Chamber IV; 17-culture solution tube; 18-trachea; 19-baffle; 20-elastic buffer film; 21-culture; 22-spring; 23-chamber body; 24-chamber cover; Open slot; 27-drive pump; 28-PLC control system; 29-information display system; 30-multifunctional connection structure I; 31-multifunctional connection structure II; 32-multifunctional connection structure III.

detailed description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the preferred embodiments are only for illustrating the present invention, but not for limiting the protection scope of the present invention.

Embodiment 1, in vitro cell and organ 3-D culture device

As shown in Figures 1-5, it includes a culture room 1, a 3-D motion shaker 2, and a culture fluid exchange system and a gas exchange system that are respectively connected to the culture room 1. Amniotic fluid development and growth environment, including the temperature environment, oxygen environment and dynamic environment of various complex movements that are compatible with the human placenta. The culture room 1 is used to simulate the placenta, the culture medium is used to simulate the amniotic fluid in the mother's placenta, and the gas exchange system is used to realize the temperature. Environment, oxygen environment, using 3-D motion shaker 2 to simulate the human compound movement of the fetus in the process of amniotic fluid development in the mother's placenta, and finally realized the cultivation environment: the temperature is 37°C; the humidity is 95-98%; the volume fraction is 5 Air with % CO ₂ ; acid-base pH value is 7.2-7.4; including fixing the culture chamber 1 in the 3-D motion shaking box 2 through elastic parts which can be flexibly disassembled, and performing various irregular movements to realize a dynamic environment .

In this embodiment, a 3-D motion shaking box is set to simulate the compound movement of the human body during the development of the fetus in the mother's placenta and amniotic fluid, and the simple harmonic motion of the suspension of the organs in the human body is simulated by elastically setting the training room in the shaking box , more realistically simulates the real growth environment of human cells or organs in the human body, improves the differentiation of various seed stem cells into mature cell density levels and effectively promotes the efficiency of 3-D cell culture, and provides a simple and economical method for the growth of in vitro organs , mild, three-dimensional, nutritionally comprehensive growth environment, accelerate cell regeneration and organ formation, compared with the prior art, the device has obvious cost advantages and popularization value.

As an improvement of this embodiment, it also includes a multi-degree-of-freedom motion mechanism arranged on one side or both sides of the 3-D motion shaker. This embodiment is arranged on both sides of the 3-D motion shaker 2. The multi-degree-of-freedom The motion mechanism includes a vertical rotation mechanism, a horizontal rotation mechanism and a multifunctional connection structure arranged between them to realize the horizontal and vertical rotation of the 3-D motion shaking box 2. The culture fluid exchange system and the gas exchange system pass through The multi-functional connection structure is connected and exchanged with the outside world to realize the gas exchange with the outside world at 37°C 5% CO ₂ and the exchange of the culture medium, so as to achieve the temperature environment, oxygen environment and amniotic fluid environment suitable for the growth of cells and organs.

As an improvement of this embodiment, the longitudinal rotation mechanism includes a longitudinal rotation shaft 3 and a speed-regulating motor 14, one end of the longitudinal rotation shaft 3 is fixed to the 3-D motion shaking box 2, and the other end is connected to the speed-regulating motor 14 The main shaft is fixed, and driven by the speed-regulating motor I4, the 3-D motion shaking box 2 is rotated left and right in the longitudinal direction.

As an improvement of this embodiment, the horizontal rotation mechanism includes a horizontal rotation frame 5, a horizontal rotation shaft 6 and a speed regulating motor II 7, one end of the horizontal rotation frame 5 is rotationally connected with one end of the longitudinal rotation shaft 3, and the other end is connected with the vertical rotation shaft 3. One end of the horizontal rotating shaft 6 is rotationally connected, and the other end of the horizontal rotating shaft 6 is fixed to the main shaft of the speed-regulating motor II 7. Driven by the speed-regulating motor II 7, the horizontal rotating shaft 6 and the horizontal rotating frame 5 are driven to rotate. Finally, the 3-D motion shaker 2 is driven to rotate left and right in the horizontal direction.

As an improvement of this embodiment, the multi-degree-of-freedom motion mechanism also includes a longitudinal driven shaft 8 fixedly arranged on the other side of the 3-D motion shaker 2, between the longitudinal driven shaft 8 and the horizontal rotating frame 5 A multifunctional connection structure I 30 is provided, a multifunctional connection structure II 31 is provided at the connection between the horizontal rotation frame 5 and the longitudinal rotation shaft 3, and a multifunctional connection structure is provided at the connection between the horizontal rotation frame 5 and the horizontal rotation shaft 6 III 32, the multifunctional connection structure includes a movable sleeve 9 that is rotatable and hermetically sleeved on the outer surface of the longitudinal rotation shaft 3 or the horizontal rotation shaft 6, and is arranged between the movable sleeve 9 and the longitudinal rotation shaft 3 or the horizontal rotation shaft 6 The sealing cavity, the rolling bearing 10 arranged between the movable sleeve 9 and the longitudinal rotating shaft 3 or the horizontal rotating shaft 6, and the rolling bearings 10 arranged in the horizontal rotating shaft 6, the horizontal rotating frame 6, the longitudinal rotating shaft 3 and the longitudinal driven shaft 8 The culture fluid circulation channel 11 and the gas exchange channel 12, the sealed cavity communicates with the culture chamber 1 through the culture fluid circulation channel 11 and the gas exchange channel 12 respectively, the sealed cavity is arranged in an annular structure, and the multifunctional connecting structure III 32 has Two mutually independent sealed chambers I, II (13, 14), the multifunctional connecting structure I 30 has a sealed chamber III 15, the multifunctional connecting structure II 31 has a sealed chamber IV 16, and the culture fluid exchange system includes successively Connected sealing chamber I 13, horizontal rotation shaft and culture solution circulation channel 11 of the horizontal rotating frame part, sealing chamber III 15, culture solution circulation channel 11 of the longitudinal driven shaft part, culture solution pipe 17 communicating with the culture solution circulation channel , the sealed chamber I 13 is communicated with the external culture solution source, and the culture solution pipe 17 is communicated with the culture chamber 1 through the 3-D motion shaking box 2; the gas exchange system includes the sealed chamber II 14, the horizontal The gas exchange channel 12 of the rotating shaft and the horizontal rotating frame part, the sealed chamber IV 16, the gas exchange channel 12 of the longitudinal rotating shaft part, and the gas pipe 18 communicated with the gas exchange channel 12. The _CO gas source is connected, and the air pipe 18 is connected with the culture chamber 1 through the 3-D motion shaking box 2 .

As an improvement of this embodiment, the above-mentioned culture fluid exchange system and gas exchange system can adjust the specific passages according to the needs, and select the appropriate sealed chamber I, sealed chamber II, sealed chamber III and sealed chamber IV.

As an improvement of this embodiment, a plurality of baffles 19 made of elastic material are evenly spaced in the culture chamber 1, which can be silica gel, etc., and the head of the baffle 19 is provided with an elastic buffer film with a circular top 20. Under the action of the baffle 19, the culture 21 can perform various irregular movements, simulating more complicated human body movements, and can form a support for the culture 21 to realize a suspended growth state; evenly spaced to a suitable density, can The chorion in the simulated maternal placenta protects the culture 21, which can avoid damage to the culture 20. In addition, the elastic buffer film 20 with a circular arc top can effectively reduce the contact area with the culture 21, which is beneficial to the culture medium. sufficient contact to promote its good growth.

As an improvement of this embodiment, the elastic member is a spring or rubber or other elastic members. The preferred spring 22 in this embodiment can perform simple harmonic vibration or swing under the action of the 3-D motion shaker, simulating the compounding during suspension. sports.

As an improvement of this embodiment, the culture chamber 1 includes a chamber body 23 and a chamber cover 24 that can seal and fix the chamber body 23. The chamber cover 24 is provided with a snap-fit structure that fits with the chamber body 23. The chamber body is provided with a snap-fit structure that matches the snap-fit structure. The snap-fit structure in this embodiment is a protrusion 25 provided on the chamber cover, and the snap-fit structure is an open groove that closely fits with the protrusion 25 26. After the culture is packed into the cultivation chamber during use, the projection of the chamber cover is used to seal in the opening groove 26, and then the two are fixed with bolts or screws or a quick-change mechanism.

As an improvement of this embodiment, a drive pump 27 is provided between the sealed chamber I and the external culture solution source, and between the sealed chamber II and the external 37° C. 5% CO ₂ gas source.

As an improvement of this embodiment, it also includes a PLC control system 28 and an information display system 29 for fully automatic control of the cultivation device, which is beneficial to monitor and manage the entire cultivation process and improve its cultivation efficiency and quality.

Example 2, the method of cultivating an artificial liver with organ structure and functionality using a 3-D culture device for cells and organs in vitro

As shown in Figure 6. The device and kit materials used in this embodiment are as follows: in vitro cell and organ 3-D culture device (3-D motion shaker with a rotating speed of 0-50rmp/min; 37°C, 5% CO ₂ culture room), conditioned medium (CM1, CM2, CM3), 100U/mL penicillin, 100μg/mL streptomycin (Cellgro, cat.no.30-001-CI), 5000U/mL penicillin, 5000μg/mL streptomycin, DMEM/F12 (Gibco -BRL, cat.no.11330), VEGF (Pepnotech, cat.no.100-20C), HGF (R&D, cat.no.294-HG-005/CF), bFGF (Pepnotech, cat.no.100- 188), bovine insulin (Sigma, cat.no.I6634), human transferrin (Sigma, cat.no.T1147), levothyroxine (Sigma, cat.no.T6397), sodium selenite (Sigma, cat. .no.S9133), putrescine (Sigma, cat.no.P5780), progesterone (Sigma, cat.no.7556), Oncostatin M, dexamethasone, non-essential amino acids, L-glutamine.

Prepare three conditioned media (Conditioned medias, CMs), respectively CM1 medium, CM2 medium, CM3 medium, the specific steps are as follows:

1) Depletion of Stem and Progenitor Cells in Embryonic Mouse Liver:

a. Under a dissecting microscope, take out the liver-like tissues of embryonic mice (E7-E15) from 7 to 15 days, five embryonic mice in each period, mix them well, grind them slowly with a homogenizer, filter, and collect the filtrate;

b. Freeze and thaw the filtrate three times under the conditions of -80°C and 39°C, 30mim each time, and then filter with a 0.45μm filter membrane to collect the filtrate;

c. Detect OX43 and OX44 positive hematopoietic stem cells and Thy-1 positive liver embryonic liver cells in the filtrate by immunofluorescence method. If the test result is negative, it shows that the stem cells and progenitor cells in the liver of the embryonic mouse have been removed;

2) Use a protein kit to detect protein content (50-100mg/mL)

3) Prepare CM1, CM2 and CM3 medium

CM1 medium: add VEGF to the mixed solution (1:4) of embryonic mouse liver filtrate and DMEM complete culture solution (1:4) from which stem cells and progenitor cells have been removed to a final concentration of VEGF of 20ng/ml;

CM2 medium: Add HGF, bFGF, bovine insulin, human transferrin, levothyroxine, and selenite to a mixed solution (1:4) of embryonic mouse liver filtrate and DMEM complete culture medium from which stem cells and progenitor cells have been removed Sodium phosphate, putrescine, progesterone until the final concentration of HGF is 10ng/mL, the final concentration of bFGF is 5ng/mL, the final concentration of bovine insulin is 0.5mg/mL, the final concentration of human transferrin is 0.5mg/mL, and the final concentration of levothyroxine The final concentration is 40ug/mL, the final concentration of sodium selenite is 34ug/mL, the final concentration of putrescine is 0.5ug/mL, and the final concentration of progesterone is 6ug/mL (please add the concentration of each component);

CM3 medium: add HGF, bFGF, Oncostatin M, dexamethasone, non-essential amino acids, and L-glucose to a mixed solution (1:4) of embryonic mouse liver filtrate and DMEM complete culture medium from which stem cells and progenitor cells have been removed Aminoamide to a final concentration of HGF of 100 ng/mL, a final concentration of bFGF of 50 ng/mL, a final concentration of Oncostatin M of 20 ng/ml, a final concentration of dexamethasone of 0.1 μM, a mass fraction of non-essential amino acids of 1% and L-glutamine The final amide concentration was 5 mM.

Then use the prepared CM1, CM2 and CM3 medium to culture in vitro 3-D artificial liver with organ structure and function, the specific method is as follows:

First, under the monitoring of the computer system, slowly inject cells including seeded stem cells into the scaffold from the portal vein and inferior vena cava using a three-step method, at least 3-5×10 ⁵ each time, 2-3 times at intervals of 15-30 minutes; then Put the WDLS injected with seed stem cells (hereinafter referred to as WDLS-NG2 ⁺ HSC) into the culture chamber, and hang the culture chamber elastically in the 3-D motion shaker, start the culture fluid exchange system and gas exchange system, and add 500 μl 3-D culture in vitro with CM1 medium for one week (1st to 7th day), start the horizontal rotation mechanism during the day, first rotate 360° to the left horizontally for 8 hours, then rotate 360° to the right horizontally for 8 hours, and start the vertical rotation mechanism and horizontal rotation at night Institutions, combined vertical and horizontal rotation combined 3-D rotation for 8 hours, supplemented with 200 μL CM1 medium every two days; after the second week of culture (8th day), replaced 500 μL CM2 medium, and the culture method and conditions were the same as the first The same for one week (day 8-14); after the third week of culture (day 15), replace 500 μL CM3 medium, the culture method and condition are the same as the first week, the dosage and method are the same as the first week, and last for 7 days. days (8-14 days); after the third week of culture (day 15), replace 500 μL of CM3 medium with the same dose and method as the first week (days 15-21), and terminate the culture after 21 days of culture to obtain Organ structure and function of the artificial liver, and then observe the histological structure under white light and fluorescence microscopes, and analyze the supernatant liver functional proteins. The results showed that a lobe-like outline of the liver was formed by day 9 (Figure 6A), and a whole liver-like outline was formed by day 21 (Figure 6B), H&E staining and culture supernatant functional Protein analysis showed that the culture had a structure similar to that of normal liver tissue (Fig. 6Ca) (Fig. 6Cb). If the central vein (the central vein, CV) of the hepatic lobule was formed (indicated by the arrow), with the prolongation of the culture time, the culture on the The secretion of serum albumin (Alb means hepatic albumin) increased (Fig. 6Cc), indicating that the seed stem cells can form an artificial liver with organ structure and function in a specific culture microenvironment, showing the seed cells with great potential for clinical application.

Finally, two points are explained: (1) The preparation of the three conditional media (CM1, CM2, CM3—) we developed is based on the theoretical basis of mammalian liver development and maturation (see Figure 7), and a lot of time has been spent on scientific research Obtained by proof, relevant articles will be published soon; (2) the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit, although the present invention has been described in detail by the above preferred embodiments, those skilled in the art It should be understood that various changes in form and details may be made therein without departing from the scope of the invention defined by the appended claims.

Claims (12)

1. The 3-D culture device in vitro of cells and organs is characterized in that: it comprises a culture chamber, a 3-D motion shaking box capable of compound motion, and a conditional culture solution circulation system and a gas exchange system communicated with the culture chamber respectively. The culture chamber is detachably suspended in the 3-D motion shaker through elastic components, and also includes a multi-degree-of-freedom motion mechanism arranged on one side or both sides of the 3-D motion shaker; The multi-degree-of-freedom motion mechanism includes a longitudinal rotation mechanism, a horizontal rotation mechanism and a connection structure arranged between them. The connection structure is mainly composed of a movable sleeve and a rolling bearing. The longitudinal rotation mechanism and the horizontal rotation mechanism are respectively passed through the rolling bearing. Connected with the movable sleeve, the movable sleeve is also provided with a sealed cavity, and the conditioned culture fluid circulation system and the gas exchange system are connected to the outside through the sealed cavity in the connection structure to exchange the culture fluid and gas. 2. The in vitro 3-D culture device for cells and organs according to claim 1, characterized in that: the culture environment of the culture chamber: the temperature is 37°C; the humidity is 95-98%; the volume fraction is 5% CO ₂ air; the acid-base pH value is 7.2-7.4. 3. The cell and organ in vitro 3-D culture device according to claim 1, characterized in that: the longitudinal rotation mechanism comprises a longitudinal rotation shaft and a speed-regulating motor 1, and one end of the longitudinal rotation shaft is connected to the 3-D motion The shaking box is fixed, and the other end is fixed with the main shaft of speed-regulating motor 1. 4. The 3-D culture device for cells and organs in vitro according to claim 3, characterized in that: the horizontal rotation mechanism comprises a horizontal rotation frame, a horizontal rotation shaft and a speed-regulating motor II, and one end of the horizontal rotation frame is connected to One end of the longitudinal rotating shaft is rotatably connected, and the other end is rotatably connected to one end of the horizontal rotating shaft, and the other end of the horizontal rotating shaft is fixed to the main shaft of the speed-regulating motor II. 5 . The in vitro 3-D culture device for cells and organs according to claim 4 , wherein connection structures are provided at the junctions between the horizontal rotation frame and the longitudinal and horizontal rotation axes. 6 . 6. The cell and organ in vitro 3-D culture device according to claim 5, characterized in that: the multi-degree-of-freedom motion mechanism is arranged on both sides of the 3-D motion shaking box, and the multi-degree-of-freedom motion mechanism also It includes a longitudinal driven shaft fixedly arranged on the other side of the 3-D motion shaker, and a connecting structure is arranged between the longitudinal driven shaft and the horizontal rotating frame. 7. The in vitro 3-D culture device for cells and organs according to claim 6, characterized in that: the connecting structure includes a rotatable outer circular surface that is sealed and fitted on the longitudinal rotating shaft or the horizontal rotating shaft or the longitudinal driven shaft The movable sleeve, the sealing cavity arranged between the movable sleeve and the longitudinal rotating shaft or the horizontal rotating shaft or the longitudinal driven shaft, the rolling bearing arranged between the movable sleeve and the longitudinal rotating shaft or the horizontal rotating shaft or the longitudinal driven shaft, and Conditioned culture solution circulation channel and gas exchange channel arranged in the horizontal rotation shaft, horizontal rotation frame, longitudinal rotation shaft and longitudinal driven shaft, and the sealed cavity communicates with the culture chamber respectively through the conditional culture solution circulation channel and gas exchange channel. 8. The 3-D culture device for cells and organs in vitro according to claim 1, characterized in that: a plurality of baffles made of elastic materials are evenly spaced in the culture chamber, and the head of the baffle is provided with Elastic cushioning membrane at the top of the arc. 9. The in vitro 3-D culture device for cells and organs according to claim 1, wherein the elastic member is a spring or rubber or other elastic members. 10. The cell and organ in vitro 3-D culture device according to claim 1, characterized in that: the culture chamber includes a chamber body and a chamber cover that can seal and fix the chamber body, and the chamber cover is provided with a Interlocking engaging structures, the chamber body is provided with an engaging engaging structure matching the engaging structures. 11. The in vitro 3-D culture device for cells and organs according to any one of claims 1-10, further comprising a PLC control system and an information display system for fully automatic control of the culture device. 12. The method for cultivating an artificial liver using the cell and organ in vitro 3-D culture device according to any one of claims 1-11, characterized in that: using the cell and organ in vitro 3-D culture device to simulate the dynamics of artificial liver growth and development Environmental cultivation of the artificial liver; the dynamic environment for the growth and development of the artificial liver includes: elastically suspending the natural decellularized whole organ scaffold of the seed cells in a 3-D culture device, at 37°C, with a volume fraction of 5% CO ₂ The next stage is cultivated, the first stage is 1 to 7 days, the second stage is 8 to 14 days, and the third stage is 15 to 21 days. 24h dynamic culture is adopted. During the day, it first rotates horizontally to the left 360°, and then horizontally rotates to the right 360° At night, the composite 3-D rotation combining vertical rotation and horizontal rotation is adopted. The rotation speed is 20-40r/min during the day and less than 20r/min at night. The medium is changed every three days, and different mediums are replaced at each stage.