CN112852627A - Method for co-culturing pluripotent stem cells from human liver and pancreatic islets based on multi-organ chip - Google Patents
Method for co-culturing pluripotent stem cells from human liver and pancreatic islets based on multi-organ chip Download PDFInfo
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Abstract
The invention relates to a method for co-culturing human liver and pancreatic islet from pluripotent stem cells based on a multi-organ chip. The multi-organ culture chip can be composed of two or more cell culture chambers, the bottoms of the cell culture chambers are provided with micro-pit (column) structures which are arranged in an array manner, so that cells can grow in a three-dimensional manner, a plurality of channels with unlimited lengths and micron-sized widths are connected in series between the cell culture chambers, and a closed continuous perfusion culture system is realized through a peristaltic pump; the liver and the pancreatic islet used for co-culture are human liver and pancreatic islet like with the induced three-dimensional tissue level by utilizing the pluripotent stem cells, have partial physiological functions of the liver and the pancreatic islet through the identification of gene, protein and secretion levels, and in the same system, the pancreatic islet cells and the liver cells are mutually promoted by the secreted cell factors, so that the co-culture of the pancreatic islet and the liver in vitro and the glucose steady-state regulation which is closer to the physiological level are realized.
Description
Technical Field
The invention relates to the field of stem cell design and tissue engineering, in particular to a multi-organ chip for co-culture of human liver and pancreatic islet from pluripotent stem cells, and particularly relates to a method for constructing a co-culture system blood sugar of liver and pancreatic islet organs from stem cells based on an organ chip technology.
Background
In recent years, the incidence of diabetes is rapidly increased, and the third chronic disease in the world seriously threatens social health. Diabetes can be classified into type 1 diabetes caused by insulin deficiency and type 2 diabetes caused by insulin resistance according to their pathogenesis. Elevated blood glucose caused by imbalanced in vivo glucose regulation causes a series of diabetic syndromes that severely affect human health. At present, pancreas or islet transplantation is the main method for fundamentally treating diabetes, but due to the problems of lack of donor quantity, immune rejection reaction after transplantation and the like, the large-scale application of the method in clinical treatment is limited. The problem is expected to be fundamentally solved based on the development of stem cell regeneration medical technology. The somatic cells from the patient are dedifferentiated into pluripotent stem cells, and the pluripotent stem cells are directionally induced into islet cells, so that the problem of cell source tension can be solved, and immune rejection after xenotransplantation can be effectively avoided.
In recent years, the rapid development of stem cell technology has led to the development of organoid models based on IPS cells, which mimic the development and formation of organs, and which can direct the development of three germ layers and contain a variety of cellular components. These all provide new approaches to disease treatment, drug screening and regenerative medicine. At present, the induced pluripotent stem cells can induce and generate normal cells such as cardiac muscle, nerve and liver in vitro, and can generate specific cell types by taking patient somatic cells as sources. However, there are few clinical treatments based on this technique, and the main factors are low differentiation levels of stem cell-derived cells, immature stages, and low differentiation purity. Therefore, the problem to be solved is to find a method for promoting islet cells to be more mature and have higher purity. It is shown that under physiological conditions, there is a close relationship between cells and tissues, and the cell secreted factors can promote the function of adjacent cells. During normal human embryonic development, liver and pancreatic island are derived from endoderm, and the liver and pancreatic island are in close and inseparable connection in function, and GLP-1 secreted by liver cells can promote pancreatic island development and secretion function. However, the research results show that the simple mixed culture of liver and islet cells has great limitation on the research of simple islets and liver functions, and the traditional two-dimensional co-culture of Transwell and the like has difficulty in constructing three-dimensional multi-component liver and islet organs. Therefore, a three-dimensional non-contact multi-organ-chip co-culture system with high flux, low cost and simple operation is continuously developed.
The organ chip technology is rapidly developed, and can combine the biophysical and chemical factors such as three-dimensional cell matrix, fluid shear force, oxygen concentration gradient and the like in multiple spatial angles. Micro-organ models with good functions such as lung, cardiac muscle, liver and the like are constructed on the chip. How to construct a culture medium and a culture environment for multi-organ co-culture is a significant challenge facing the traditional stem cell source tissue-like organ at present. How to establish a liver and pancreatic islet model with good in-vitro functions by means of a multi-organ chip technology so as to obtain large-scale and stable pancreatic islet cells for treatment and drug evaluation of diabetes, and basic research is the key point of the invention.
Disclosure of Invention
The invention aims to provide a multi-organ chip for co-culture of human liver and pancreatic islets from pluripotent stem cells, in the system, pancreatic islet cells and liver cells are mutually promoted by secreted cytokines, and the long-term in-vitro co-culture of pancreatic islets and liver and the glucose steady-state regulation closer to the physiological level are realized. Meanwhile, the activity and secretion function of the pancreatic island and the hepatocyte are more favorable in a co-culture system.
In order to achieve the purpose, the invention adopts the technical scheme that:
the multi-organ culture chip can be composed of two or more cell culture chambers, the bottoms of the cell culture chambers are provided with micro-pit (column) structures which are arranged in an array manner, so that cells can grow in a three-dimensional manner, a plurality of channels with unlimited lengths and micron-sized widths are connected in series between the cell culture chambers, and a closed continuous perfusion culture system is realized through a peristaltic pump; the liver and the pancreatic island used for co-culture are human liver and pancreatic island-like by utilizing the induced three-dimensional tissue level of the pluripotent stem cells, have partial physiological functions of the liver and the pancreatic island through the identification of gene, protein and secretion level, and in the same system, the pancreatic island cells and the liver cells are mutually promoted by the separated cell secretion factors, thereby realizing the co-culture of the pancreatic island and the liver in vitro and the glucose steady state regulation closer to the physiological level. Meanwhile, the activity and secretion function of the pancreatic island and the hepatocyte are more favorable in a co-culture system. The invention constructs a liver-like and islet tissue co-culture organ chip system derived from in vitro pluripotent stem cells for the first time, and provides an in vitro model for the alternative treatment of diabetes, disease research and drug development.
The invention provides a multi-organ chip, which comprises two or more than two cell culture chambers, wherein the bottom of each culture chamber is provided with a micro-array structure, namely a micro-pit (column) structure in array arrangement; a plurality of channels with unlimited length and micron-sized width are connected in series between the cell culture chambers, and a closed continuous perfusion culture system is realized through a peristaltic pump.
The multi-organ chip comprises an upper-layer structure PDMS 1, a lower-layer structure PDMS 2, a cell inlet pool (3; 4; 5; 6; 7; 8; 9; 10), a cell culture chamber connecting channel 11, and a cell culture chamber (12; 13; 14; 15; 16; 17; 18; 19); is connected with the peristaltic pump through a perfusion tube 20 and a port valve 21;
wherein the cell inlet reservoir (3; 4; 5; 6) is communicated with the cell culture chamber (12; 13; 14; 15), the cell culture chamber (12; 13; 14; 15) is communicated with the cell culture chamber (16; 17; 18; 19) through the cell culture chamber connecting channel 11, and the cell culture chamber (16; 17; 18; 19) is communicated with the cell inlet reservoir (7; 8; 9; 10).
The cell inlet pool 3 is connected with a perfusion tube 20 through a connecting port 21 for injecting cell culture medium; the cell inlet pool 7 is communicated with the cell inlet pool 8 through an perfusion tube; the cell inlet pool 4 is communicated with the cell inlet pool 5 through an perfusion tube; the cell inlet pool 9 is connected with the cell inlet pool 10 through a perfusion tube, and the cell inlet pool 6 is connected with the perfusion tube through a connecting port to output cell culture medium; the culture medium injection pipe and the culture medium output pipe are arranged in the same culture medium storage bottle to form a closed perfusion system for perfusion of the multi-organ chip.
The upper layer structure of the cell culture chamber has the length of 5-10 mm, the width of 3-6 mm and the height of 0.5-2 mm; the depth of the lower layer microarray structure is 0.4-1.5 mm, and the radius is 0.2-0.4 mm; the height of the micro-channel (11) is 0.1 mm, the height is 0.1 mm, the length is 0.5-5 mm, and the number of arrays is 10-20.
The number of the lower layer microarray structures in the cell culture chamber is 40-200.
The lower micro-pit array structure of the cell culture chamber can be a micro-column structure with the height of 0.4-1.5 mm and the radius of 0.2-0.4 mm.
Flow rates of medium used for multi-organ chip perfusion were 10-20 microliters per minute.
The invention provides a method for co-culturing human liver and pancreatic islets from pluripotent stem cells based on the multi-organ chip, wherein the liver and pancreatic islets for co-culturing are human liver-like and pancreatic islets at a three-dimensional tissue level by utilizing the induction of the pluripotent stem cells, partial physiological functions of the liver and the pancreatic islets are identified through gene, protein and secretion levels, and in the same system, pancreatic islet cells and liver cells are mutually promoted through secreted cytokines, so that the co-culturing of the pancreatic islets and the liver in vitro and the glucose steady-state regulation closer to the physiological level are realized.
The tissue-like size for co-culture of liver and pancreatic islets is 0.2-0.5 mm.
The medium composition used for co-cultivation was DF12 supplemented with 1% B27, 1% N2, 1% non-essential amino acids, 1% GLUTAMAX to a final concentration of 10mM glucose.
The chip comprises an upper layer with a structure PDMS 1 and a lower layer with a structure PDMS 2, and consists of cell inlet tanks 3, 4, 5, 6, 7, 8, 9 and 10, a cell culture chamber connecting channel 11, cell culture chambers 12, 13, 14, 15, 16, 17, 18 and 19, and is connected with a peristaltic pump through an perfusion tube 20 and an interface valve 21. Wherein, the cell inlet pool 3 is communicated with the cell culture chamber 12, the cell culture chamber 12 is communicated with the cell culture chamber 16 through the cell culture chamber connecting channel 11, and the cell culture chamber 16 is communicated with the cell inlet pool 7; the cell inlet pool 4 is communicated with a cell culture chamber 13, the cell culture chamber 13 is communicated with a cell culture chamber 17 through a cell culture chamber connecting channel, and the cell culture chamber 17 is communicated with the cell inlet pool 8; the cell inlet pool 5 is communicated with the cell culture chamber 14, the cell culture chamber 14 is communicated with the cell culture chamber 18 through a cell culture chamber connecting channel, and the cell culture chamber 18 is communicated with the cell inlet pool 9; the cell inlet pool 6 is communicated with the cell culture chamber 15, the cell culture chamber 15 is communicated with the cell culture chamber 19 through a cell culture chamber connecting channel, and the cell culture chamber 19 is communicated with the cell inlet pool 10. After the inoculation with cell balls, the cell inlet tank 3 is connected to a perfusion tube 20 through a connection port 21, and a cell culture medium is injected. The cell inlet pool 7 is communicated with the cell inlet pool 8 through an perfusion tube; the cell inlet pool 4 is communicated with the cell inlet pool 5 through an perfusion tube; the cell inlet pool 9 is connected with the cell inlet pool 10 through a perfusion tube; the cell inlet pool 6 is connected with a perfusion tube through a connecting port to output cell culture medium. The culture medium injection pipe and the culture medium output pipe are arranged in the same culture medium storage bottle to form a closed perfusion system.
The upper layer structure of the cell culture chamber (3-10) is 5-10 mm long, 3-6 mm wide and 0.5-2 mm high, the depth of the lower layer microarray structure of the cell culture chamber (3-10) is 0.4-1.5 mm, and the radius is 0.2-0.4 mm.
The number of the lower layer microarray structures of the cell culture chamber (3-10) is 40-200.
The height of the micro-channel 11 is 0.1 mm, the height is 0.1 mm, the length is 0.5-5 mm, and the number of arrays is 10-20.
The multi-organ chip for co-culture of the pluripotent stem cells from the human liver and the pancreatic islets is characterized in that: the lower layer micro-pit array structure of the cell culture chamber (3-10) can be changed into a micro-column structure with the height of 0.4-1.5 mm and the radius of 0.2-0.4 mm.
The multi-organ chip for co-culture of the pluripotent stem cells from the human liver and the pancreatic islets is characterized in that: pluripotent stem cells used for inducing liver and pancreatic islets include embryonic stem cells and induced pluripotent stem cells.
In the islet and liver co-culture system, the insulin secretion amount of the islets in the co-culture system is obviously improved compared with that of the islets cultured independently through detection.
In the islet and liver co-culture system, the albumin secretion amount of the liver in the co-culture system is obviously improved by detection compared with that of the liver cultured independently.
In the islet and liver co-culture system, the islet specific markers PDX1, NKX6.1, GCG, SST and PPY related genes and protein expression in the co-culture system are obviously improved through identification.
In the islet and liver co-culture system, the expression of liver specific markers ALB, CYP3A4 and CK7 related genes and proteins in the co-culture system is obviously improved through identification.
In the islet and liver co-culture system, the expression of islet function-related specific markers E-cadherin, Glut-1-related genes and proteins in the co-culture system is remarkably improved through identification.
In the islet and liver co-culture system, the liver function related specific markers INSR and Glut-4 in the co-culture system are identified, and the expression of related genes and proteins is obviously improved.
In the islet and liver co-culture system and the GTT and ITT detection co-culture system, the regulation and control of sugar steady state and insulin sugar stimulation reaction capability are improved, and the in vivo reaction capability is more similar.
The multi-organ chip for co-culture of the pluripotent stem cells from the human liver and the pancreatic islets is characterized in that: the tissue-like size for co-culture of liver and pancreatic islets is 0.2-0.5 mm.
The multi-organ chip for co-culture of the pluripotent stem cells from the human liver and the pancreatic islets is characterized in that: the medium composition used for co-cultivation was DF12 supplemented with 1% B27, 1% N2, 1% non-essential amino acids, 1% GLUTAMAX to a final concentration of 10mM glucose.
The multi-organ chip for co-culture of the pluripotent stem cells from the human liver and the pancreatic islets is characterized in that: flow rates of medium used for multi-organ chip perfusion were 10-20 microliters per minute.
The invention has the following advantages:
1. the multi-organ three-dimensional co-culture system designed by the invention is suitable for co-culture of other three-dimensional organoids, and is a multi-organ co-culture system with wide application prospect and simple operation.
2. Can well simulate the physiological environment in vivo and is beneficial to maintaining the functions of cells in vitro. On one hand, the multi-organ co-culture system can promote the interaction between organs, realize the mutual promotion of cell functions and the long-term maintenance of in vitro functions. On the other hand, the three-dimensional co-culture perfusion system simulates the physical microenvironment in vivo, provides the fluid shear force close to the physiological condition, and realizes the high activity and high function growth of cells.
3. The high-flux three-dimensional co-culture system can realize dynamic large-scale in-vitro co-culture of the liver and the pancreatic island. Due to the technical advantages of high flux, high integration, simple operation and less consumption of cell culture medium in the microfluidic technology, the in-vitro large-scale co-culture of the liver and the pancreatic islet can be realized, and the research requirements of medicines are met.
4. Can realize the separation of the islet and the liver cell in the co-culture system and can realize the operation of a single type of cell. The two cell culture chambers of the co-culture chip are connected by a micron-sized channel, when the pancreatic islets or the liver tissues in a single culture chamber are treated, due to size limitation, cell clusters cannot enter the other cell culture chamber, a pipette can be repeatedly used for blowing and beating to obtain single pancreatic islets or liver tissues, and the damage to the cells caused by chemical or enzyme treatment is avoided.
5. Has wide application prospect. With the rapid development of the microfluidic technology and the organoid technology, the invention combines and applies the two technologies, realizes the establishment of the islet and liver organoid co-culture system of two stem cell sources in vitro, and provides a powerful platform for islet transplantation treatment of diabetes, research of related diseases and drug evaluation.
Drawings
Fig. 1 is a schematic diagram of the whole structure of the microfluidic chip of the present invention, wherein the upper layer has a structure PDMS (1), the lower layer has a structure PDMS (2), cell inlet reservoirs (3), (4), (5), (6), (7), (8), (9), (10), cell culture chamber connecting channels (11), cell culture chambers (12), (13), (14), (15), (16), (17), (18), (19), and perfusion tubes (20) interface valves (21).
FIG. 2 is a diagram showing the development and identification of liver organoids derived from HiPSCs;
FIG. 3 is a diagram showing the development and identification of islet organoids derived from HiPSCs;
FIG. 4 measurement of hepatic and islet glucose metabolism and secretion function under single culture and co-culture conditions.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
The multi-inducible stem cell source human liver and islet organoid co-culture chip is constructed by utilizing a micro-fluidic chip which is designed and manufactured by a laboratory, and the configuration is shown in figure 1. The micro-fluidic chip mainly comprises a micro-fluidic chip which comprises an upper layer with a structure PDMS 1 and a lower layer with a structure PDMS 2, cell inlet cells 3, 4, 5, 6, 7, 8, 9 and 10, a cell culture chamber connecting channel 11, cell culture chambers 12, 13, 14, 15, 16, 17, 18 and 19, and is connected with a peristaltic pump through a perfusion tube 20 and an interface valve 21. In the co-culture experiment, induced islet cell microspheres were resuspended in 5 x 10 medium containing DF12 supplemented with 1% B27, 1% N2, 1% non-essential amino acids, 1% GLUTAMAX, and 10mM glucose final concentration3Inoculating the microspheres into cell culture chambers 3, 8, 5 and 10 at a density of per milliliter, treating the microspheres of liver organoids in the same way, inoculating the microspheres into cells 4, 6, 7 and 9, connecting the cell inlet pool 3 with a perfusion tube 20 through a connecting port 21 to inject cell culture medium after the cells naturally settle, and connecting the cell inlet pool 6 with the perfusion tube through the connecting port to output the cell culture medium. Only liver tissue or islet tissue culture medium injection pipe and culture medium output pipe are added to all small chambers of the control group in the same culture medium storage bottle to form a closed perfusion system. The culture was performed at a perfusion rate of 10 microliters per minute. During the culture period, cell viability and function identification was performed every 5 days.
The liver and islet organoids which have been induced to differentiate are subjected to functional identification, and the results in FIG. 2 show islet organoid proteins and cell types (INS, CGC, PPY, SST) of islets identified at the gene level; FIG. 3 results show that the cell types of pancreatic islets were identified at the liver organoid protein and gene level (ALB, CK7, CYP3A 4); the results in FIG. 4 show the glucose metabolism, insulin secretion, albumin secretion and urea secretion in the co-culture system and the single culture system, and it can be seen from the data that co-culture is more advantageous for the regulation of glucose metabolism in liver and pancreatic islets, insulin secretion in pancreatic islets, and albumin and urea secretion in liver cells, and the results are more apparent after 15 days of co-culture.
Claims (10)
1. A multi-organ chip, characterized by: the multi-organ chip comprises two or more cell culture chambers, and the bottom of each culture chamber is provided with a micro-array structure, namely a micro-pit (column) structure arranged in an array; a plurality of channels with unlimited length and micron-sized width are connected in series between the cell culture chambers, and a closed continuous perfusion culture system is realized through a peristaltic pump.
2. The multi-organ-chip according to claim 1, characterized in that: the multi-organ chip comprises an upper-layer structure PDMS (1), a lower-layer structure PDMS (2), a cell inlet pool (3; 4; 5; 6; 7; 8; 9; 10), a cell culture chamber connecting channel (11), a cell culture chamber (12; 13; 14; 15; 16; 17; 18; 19); is connected with the peristaltic pump through a perfusion tube (20) and a port valve (21);
wherein the cell inlet reservoir (3; 4; 5; 6) is communicated with the cell culture chamber (12; 13; 14; 15), the cell culture chamber (12; 13; 14; 15) is communicated with the cell culture chamber (16; 17; 18; 19) through the cell culture chamber connecting channel (11), and the cell culture chamber (16; 17; 18; 19) is communicated with the cell inlet reservoir (7; 8; 9; 10).
3. The multi-organ-chip according to claim 2, characterized in that: the cell inlet pool (3) is connected with the perfusion tube (20) through a connecting port (21) to inject cell culture medium; the cell inlet pool (7) is communicated with the cell inlet pool (8) through an perfusion tube; the cell inlet pool (4) is communicated with the cell inlet pool (5) through an perfusion tube; the cell inlet pool (9) is connected with the cell inlet pool (10) through a perfusion tube, and the cell inlet pool (6) is connected with the perfusion tube through a connecting port to output cell culture medium; the culture medium injection pipe and the culture medium output pipe are arranged in the same culture medium storage bottle to form a closed perfusion system for perfusion of the multi-organ chip.
4. The multi-organ-chip according to claim 1, characterized in that: the upper layer structure of the cell culture chamber has the length of 5-10 mm, the width of 3-6 mm and the height of 0.5-2 mm; the depth of the lower layer microarray structure is 0.4-1.5 mm, and the radius is 0.2-0.4 mm; the height of the micro-channel (11) is 0.1 mm, the height is 0.1 mm, the length is 0.5-5 mm, and the number of arrays is 10-20.
5. The multi-organ-chip according to claim 1, characterized in that: the number of the lower layer microarray structures in the cell culture chamber is 40-200.
6. The multi-organ-chip according to claim 1, characterized in that: the lower micro-pit array structure of the cell culture chamber can be a micro-column structure with the height of 0.4-1.5 mm and the radius of 0.2-0.4 mm.
7. The multi-organ chip for co-culture of pluripotent stem cell-derived human liver and pancreatic islets according to claim 3, wherein: flow rates of medium used for multi-organ chip perfusion were 10-20 microliters per minute.
8. A method for co-culturing human liver and pancreatic islets derived from pluripotent stem cells based on the multi-organ chip of any one of claims 1 to 7, wherein the method comprises: the liver and the pancreatic islet used for co-culture are human liver and pancreatic islet like with three-dimensional tissue level induced by pluripotent stem cells, have partial physiological functions of the liver and the pancreatic islet through gene, protein and secretion level identification, and in the same system, pancreatic islet cells and the liver cells are mutually promoted by secreted cytokines, so that the co-culture of the pancreatic islet and the liver in vitro and the glucose steady-state regulation closer to the physiological level are realized.
9. The method of claim 8, wherein the method comprises: the tissue-like size for co-culture of liver and pancreatic islets is 0.2-0.5 mm.
10. The method of claim 8, wherein the method comprises: the medium composition used for co-cultivation was DF12 supplemented with 1% B27, 1% N2, 1% non-essential amino acids, 1% GLUTAMAX to a final concentration of 10mM glucose.
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