Human body micro-physiological system based on whole hPSC

Abstract

The invention belongs to the field of micro-physiological systems, and discloses a human body micro-physiological system based on whole hPSC. Three-dimensional modeling is carried out according to the relative positions and sizes of the organs in the human body, partitioning is carried out according to different areas of the human body, the brain chamber is located in the cranial cavity area, the heart and double lung chambers are located in the thoracic cavity area, and the liver and double kidney chambers are located in the abdominal cavity area. The lung chambers are arranged on the two sides of the heart, the liver is arranged on the right of the front upper portion of the abdominal cavity area, and the kidney chambers are arranged on the rear lower portion, the left portion and the right portion of the abdominal cavity area and distributed in a splayed mode. Due to the fact that printing of a whole human body chip cannot be achieved through an existing 3D printing technology, and openness of all cavities needs to be reserved to facilitate cell loading, the strategy that 3D printing is conducted according to modules is adopted, and nine bionic modules including a head top module, a cranial cavity module, a thoracic cavity module, an abdominal cavity module, a hip-leg module, a forearm module and a shank module are included. And by adopting a double-channel design, all organ chambers are connected together, so that the information exchange of all organs is promoted.

Description

Technical Field

[0001] The present invention belongs to the field of microphysiological systems and relates to a human microphysiological system based on fully human pluripotent stem cells (hPSCs). Background Art

[0002] Driven by the dual demands of precision medicine and drug screening, organ-on-a-chip technology is accelerating its evolution from single-organ function simulation to multi-organ system integration. Traditional drug screening relies on animal models and two-dimensional cell culture, suffering from significant species differences, low reduction of physiological microenvironment, and lack of multi-organ synergistic effects. Most traditional chips adopt a planar layout with simple splicing of functional modules, ignoring the three-dimensional spatial positions of organs such as the brain, heart, lungs, liver, and kidneys in the human body. Multi-organ system simulation can effectively solve the above problems. However, in the simulation of each organ, the simulation of the liver is a difficult point, mainly because of the difficulty in culturing hepatocytes in vitro.

[0003] In vitro culture models of hepatocytes are crucial for simulating liver physiological functions, evaluating drug metabolism and toxicity. As the main executors of liver functions, the in vitro acquisition of hepatocytes has long relied on primary hepatocytes or immortalized cell lines. However, primary hepatocytes are prone to losing their functions during culture, and immortalized cell lines have insufficient differentiation levels, making it difficult to simulate the metabolic, detoxification, and synthesis functions of natural hepatocytes. Pluripotent stem cells have the ability of unlimited proliferation and the potential to differentiate into three germ layers (endoderm, mesoderm, and ectoderm), and can differentiate into various cell types, such as the differentiation of the brain (ectoderm), heart (mesoderm), liver (endoderm), lungs (endoderm), and kidneys (mesoderm). By inducing pluripotent stem cells to differentiate into definitive endoderm, and then directing the obtained endoderm cells to differentiate into hepatocytes with liver functions, it provides a source of metabolically functional hepatocytes for the liver module in the constructed human microphysiological system. Summary of the Invention

[0004] The purpose of the present invention is to provide a human microphysiological system based on fully hPSCs. Its cell culture chambers can respectively simulate the parenchyma of organs such as the brain, heart, lungs, liver, and kidneys, and construct a bionic platform with interconnected multi-organs.

[0005] Technical Solution of the Present Invention

[0006] A human microphysiological system based on fully human pluripotent stem cells (hPSCs) is three-dimensionally modeled according to the relative positions and sizes of various organs in the human body and divided into zones according to different regions of the human body (cranial cavity zone, thoracic cavity zone, abdominal cavity zone). Among them, the brain chamber is in the cranial cavity zone, the heart and bilateral lung chambers are in the thoracic cavity zone, and the liver and bilateral kidney chambers are in the abdominal cavity zone. Moreover, the lung chambers are located on both sides of the heart, the liver is located in the upper front right of the abdominal cavity zone, and the kidney chambers are in the lower back of the abdominal cavity zone, one on each side, distributed in a "V" shape. Since the existing 3D printing technology cannot achieve the printing of the entire human chip and the openness of each chamber needs to be retained for convenient cell loading, a strategy of 3D printing in modules is adopted. It contains a total of 9 bionic modules, including the head top module, cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, small arm module, and lower leg module. A dual-channel design is used to connect the organ chambers to promote information exchange among the organs.

[0007] Further, a small channel is opened above the rear of each open chamber in the head top module, thoracic cavity module, abdominal cavity module, and buttock and leg module, and the small channel is perpendicular to the chamber.

[0008] Further, the lower planes of the head top module, cranial cavity module, thoracic cavity module, abdominal cavity module, and buttock and leg module all have protrusions, and at least one protrusion is provided on each plane, and the shape can be square, cylindrical, etc.

[0009] Further, the upper planes of the cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, small arm module, and lower leg module all have grooves, and at least one groove is provided on each plane, and the shape can be square, cylindrical, etc., corresponding one by one to the protrusions on the lower surfaces of the head top module, cranial cavity module, thoracic cavity module, abdominal cavity module, and buttock and leg module.

[0010] Further, the human microphysiological system based on hPSCs has a dual-channel. MH culture medium provides nutritional supply for each organ chamber through the dual-channel. Channel 1 enters from the right hand of the human body, flows through each chamber, and then flows out from the feet; Channel 2 enters from the left hand of the human body, flows through each chamber, and then flows out from the feet.

[0011] Further, the physical height of the human microphysiological system is 200 - 260 mm, the arm width is 157 - 217 mm, and the body thickness is 13 - 73 mm, and it can be scaled up or down proportionally according to height, arm width, and body thickness.

[0012] Further, the material of the human microphysiological system is one of plastic, resin, glass, quartz, silicon, ceramic, or metal.

[0013] Further, the number of chambers of the human microphysiological system is greater than or equal to 7, and the shapes, depths, and volumes of the chambers are not limited.

[0014] Further, the human microphysiological system can adopt functional cells induced and differentiated from hPSCs.

[0015] Advantages of the present invention: The present invention constructs a human microphysiological system that simulates the human anatomical structure, and by virtue of the differentiation potential of pluripotent stem cells to differentiate into endoderm, mesoderm, and ectoderm, a hepatocyte source with metabolic function is pre-differentiated. The brain, heart, lung, and kidney modules in the human microphysiological system can also adopt a similar process of hepatic differentiation, and finally, a human microphysiological system is constructed from pluripotent stem cells differentiating into brain, heart, lung, liver, and kidney cell sources with corresponding functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a modeling diagram of the human microphysiological system; among them, A is a three-dimensional modeling diagram; B is a schematic diagram of the internal structure; C is a modular partition diagram;

[0017] Figure 2 is the concave-convex structure at the segmentation of the human microphysiological system;

[0018] Figure 3 is a physical diagram of the human microphysiological system; among them, A are 9 modules printed by 3D; B is a physical diagram of the module partition;

[0019] Figure 4 is a schematic diagram of the pathway flow direction of the human microphysiological system;

[0020] Figure 5 is an immunofluorescence diagram of hepatic differentiation, scale bar: 100 μm; among them, A is the nuclear localization diagram of ALB; B is the fluorescence diagram of ALB; C is the Merge diagram of ALB; D is the nuclear localization diagram of HNF4α; E is the fluorescence diagram of HNF4α; F is the Merge diagram of HNF4α; G is the nuclear localization diagram of CK18; H is the fluorescence diagram of CK18; I is the Merge diagram of CK18;

[0021] Figure 6 is a gene expression level diagram of hepatic differentiation; among them, (A) is the relative gene expression level of CK19, a bile duct marker, during the hepatic differentiation stage, and (B) is the relative gene expression level of ALB, a hepatocyte marker, during the hepatic differentiation stage;

[0022] Figure 7 is a diagram of the secretion levels of albumin and urea nitrogen during hepatic differentiation; among them, (A) is the albumin secretion amount during the hepatic differentiation stage, and (B) is the urea secretion amount during the hepatic differentiation stage; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following further describes the specific embodiments of the present invention in conjunction with the drawings and technical solutions.

[0024] Example 1: Construction of a Human Microphysiological System

[0025] According to the relative positions and sizes of the brain, heart, lungs, liver, and kidneys in the human body, the organ chambers are partitioned according to different regions of the human body (cranial cavity region, thoracic cavity region, abdominal cavity region). Among them, the brain chamber is in the cranial cavity region, the heart and bilateral lung chambers are in the thoracic cavity region, and the liver and bilateral kidney chambers are in the abdominal cavity region. As Figure 1 shown, 3D modeling is carried out through ZBrush software. Figure A shows the front and back of the human microphysiological system model. On the back, it can be seen that thin channels (with a diameter of 1 mm) are opened in the chamber parts of each module, and a breathable membrane covers its surface, which can achieve the exchange between the inside of the chamber and the external environment and can also avoid the problem of bubbles generated by the flow of MH culture solution in the chip; Figure B shows the front and side of the internal structure of the human microphysiological system model. It can be seen that the lung chambers are located on both sides of the heart, the liver chamber is located in the upper front right position of the abdominal cavity region, and the kidney chambers are located in the lower rear position of the abdominal cavity region, one on each side, showing a "V" shape distribution; Since the existing 3D printing technology cannot achieve the printing of the entire human chip and the openness of each chamber needs to be retained to facilitate cell loading, a strategy of 3D printing in modules is adopted. Figure C shows the segmentation diagram of the human microphysiological system model, including the following bionic modules: the top of the head module, cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, forearm module, and calf module. The various modules can be adhered to form a bionic whole through buckles, threads, resin glue, etc. To enable the top of the head module, cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, forearm module, and calf module to be accurately adhered, as Figure 2 shown, there are protrusions on the lower planes of the top of the head module, cranial cavity module, thoracic cavity module, abdominal cavity module, and buttock and leg module, with at least one protrusion on each plane. There are grooves on the upper planes of the cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, forearm module, and calf module, with at least one groove on each plane, and the protrusions on the lower planes of the top of the head module, cranial cavity module, thoracic cavity module, abdominal cavity module, and buttock and leg module correspond one by one to the grooves on the upper planes of the cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, forearm module, and calf module. Combining with SLA 3D printing technology, the divided top of the head module, cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, and forearm module are printed and formed with transparent resin, and then polished and cleaned, which has the characteristics of high transparency and low water absorption rate, as Figure 3 shown as the 3D printed physical diagram of the human microphysiological system. Figure A shows the 9 printed modules, and Figure B shows the partitioned physical diagrams of the cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, forearm module, and calf module. The total height of the 3D printed human body is 230 mm, the arm width of the human body is 187 mm, and the thickness of the human body is 43 mm. This three-dimensional human microphysiological system adopts a dual-channel design to connect the organ chambers and promote the information exchange of each organ, asFigure 4 As shown, it is a schematic diagram of the passage of this double-channel (with a diameter of 1.6 mm). Channel 1 and Channel 2 in the bionic whole form two circular passages respectively. Channel 1 enters from the liquid inlet at the right hand of the human body, flows through each chamber, and then flows out from the liquid outlets 1 and 2 at the feet; Channel 2 enters from the liquid inlet 2 at the left hand of the human body, flows through each chamber, and then flows out from the liquid outlets 3 and 4 at the feet.

[0026] Example 2: Differentiation of pluripotent stem cells into hepatocytes

[0027] The program for differentiating pluripotent stem cells into hepatocytes is a process of directing the differentiation of pluripotent stem cells into hepatocytes with specific functions through specific induction conditions. Its core principle is to simulate the embryonic development process and activate specific gene expression programs by regulating extracellular signaling molecules (such as growth factors, small molecule compounds) and the microenvironment (such as Matrigel, cell density), thereby driving stem cells to gradually lose pluripotency and acquire the target cell phenotype. The differentiation of hepatocytes mainly includes three stages: the differentiation of definitive endoderm, the differentiation of hepatic progenitor cells, and the maturation of hepatocytes. First is the differentiation of definitive endoderm. Using pluripotent stem cell H9 as the source cell, it is maintained in PS medium for 1 day and in DE medium for 2 days to obtain a stable source of definitive endoderm. Then, it is cultured in FE medium for 3 days, HB medium for 4 days, and HP medium for 4 days to obtain stable hepatic progenitor cells. Finally, it is cultured in MH medium for 16 days to obtain hepatocytes with liver function. During this period, combined with immunofluorescence staining technology, real-time fluorescence quantitative PCR technology, and the detection of albumin and urea in the culture supernatant of hepatocytes, the liver function is characterized together. CK18 is cytokeratin 18, which is usually expressed in epithelial cells. As hepatocytes are cells of epithelial origin, the expression of CK18 marks its epithelial characteristics; HNF4α is hepatocyte nuclear factor 4α, which belongs to nuclear receptor transcription factors. The expression of HNF4α indicates the functional maturity of hepatocytes; ALB is the main protein synthesized by hepatocytes and is an important marker of mature hepatocytes. During the process of differentiating stem cells into hepatocytes, the expression of these three markers is usually detected to verify the maturity. First, protein characterization is performed on the cells at the stage of hepatic differentiation and maturation. As Figure 5 shown, using immunofluorescence staining technology, during the period from the 9th day to the 16th day of culture in MH medium, the markers of mature hepatocytes: HNF4α (the core of functional regulation), CK18 (structural marker), and ALB (functional maturity marker) are all positively expressed. Hepatic progenitor cells have the potential for bidirectional differentiation and can give rise to cholangiocytes or hepatocytes. SB431542 blocks the differentiation of hepatic progenitor cells into cholangiocytes. As Figure 6 shown, gene characterization is performed on the cells at the stage of hepatic differentiation and maturation. As hepatocytes mature, the expression of cholangiocyte markers such as CK19 decreases, and the expression of the hepatocyte maturation marker ALB gradually increases. At the same time, albumin and urea are detected in the supernatant at the stage of hepatic differentiation and maturation. AsFigure 7 As shown, it is the albumin and urea secretion amounts on the 9th day of culture in MH culture medium. The albumin secretion level reached 23 μg / million cells / day, and the urea secretion level reached approximately 750 μg / million cells / day.

[0028] In this experiment, by conducting a detailed study on each step of the induction and differentiation of pluripotent stem cells into hepatocytes, mature hepatocytes were finally obtained, providing a source of hepatocytes with metabolic functions for the liver module in the constructed microphysiological system.

Claims

1. A human microphysiological system based on fully hPSCs, characterized in that, Three-dimensional modeling is carried out according to the relative positions and sizes of various organs in the human body, and the human body is divided into regions, including the cranial cavity region, the thoracic cavity region, and the abdominal cavity region; among them, the cerebral ventricles are located in the cranial cavity region, the heart and the two lung cavities are located in the thoracic cavity region, and the liver and the two kidney cavities are located in the abdominal cavity region; the lung cavities are located on both sides of the heart, the liver is located in the upper front right of the abdominal cavity region, and the kidney cavities are located in the lower back of the abdominal cavity region, one on each side, showing an "eight" character distribution; a strategy of 3D printing by modules is adopted, including 9 bionic modules, including the top of the head module, the cranial cavity module, the thoracic cavity module, the abdominal cavity module, the buttock and leg module, the forearm module, and the calf module; a dual-channel design is adopted to connect the various organ cavities and promote the information exchange of the various organs.

2. The human microphysiological system based on all hPSCs according to claim 1, wherein In each of the open cavities of the cranial top module, thoracic cavity module, abdominal cavity module, and buttock and leg module, a small channel is opened above the rear of the cavity, and the small channel is perpendicular to the cavity.

3. The human microphysiological system based on all hPSCs according to claim 1, wherein The lower planes of the cranial top module, cranial cavity module, thoracic cavity module, abdominal cavity module, and buttock and leg module all have protrusions, and at least one protrusion is provided on each plane.

4. The human microphysiological system based on all hPSCs according to claim 1, characterized in that, The upper planes of the cranial cavity module, thoracic cavity module, abdominal cavity module, buttock and leg module, forearm module, and calf module all have grooves, and at least one groove is provided on each plane, corresponding one by one to the protrusions on the lower surfaces of the cranial top module, cranial cavity module, thoracic cavity module, abdominal cavity module, and buttock and leg module.

5. The human microphysiological system based on all hPSCs according to claim 1, wherein The human microphysiological system based on all hPSCs is provided with a dual-channel; MH culture medium provides nutrient supply for each organ cavity through the dual-channel. Channel 1 enters from the right hand of the human body, flows through each cavity, and then flows out from the feet; Channel 2 enters from the left hand of the human body, flows through each cavity, and then flows out from the feet.

6. The human microphysiological system based on all hPSCs according to claim 1, wherein The physical height of the human microphysiological system is 200 - 260 mm, the arm width is 157 - 217 mm, and the body thickness is 13 - 73 mm, and it is enlarged or reduced in proportion according to the height, arm width, and body thickness.

7. The human microphysiological system based on all hPSCs according to claim 1, characterized in that The material of the human microphysiological system is plastic, resin, glass, quartz, silicon, ceramic, or metal.

8. The human microphysiological system based on fully hPSCs according to claim 1, characterized in that, The number of cavities of the human microphysiological system is greater than or equal to 7, and the shapes, depths, and volumes of the cavities are not limited.

9. The human microphysiological system based on fully hPSCs according to claim 1, characterized in that, The human microphysiological system uses relevant functional cells induced by hPSCs.

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