Biomimetic vascularized ipsc-hep spheroid for liver regeneration, and preparation method therefor

Abstract

Disclosed in the present invention are a biomimetic vascularized iPSC-hep spheroid for liver regeneration, and a preparation method therefor. The biomimetic vascularized iPSC-hep spheroid is a microcapsule with a porous hyaluronic acid methacryloyl as a shell and a human-induced pluripotent stem-cell-derived hepatocyte spheroid as an inner phase, and human umbilical vein endothelial cells adhere to the surface of the shell. The biomimetic vascularized iPSC-hep spheroid of the present invention promotes effective communication between hiPSC-heps and HUVECs, and provides barrier protection for an encapsulated hiPSC-hep spheroid so as to avoid attack by the immune system. Compared with dispersed cells, the cell-to-cell interaction in the cell spheroid improves cell-to-cell communication, helps to simulate the physiological heterogeneous structure and cell microenvironment of the liver, and can more effectively repair an acutely failing liver.

Description

Biomimetic vascularized iPSC-hepatocyte spheroids for liver regeneration and methods of making the same TECHNICAL FIELD

[0001] The present invention belongs to the field of biomedical materials, in particular relates to a kind of biomimetic vascularized iPSC-hepatocyte spheroids for liver regeneration and methods of making the same. BACKGROUND

[0002] Acute liver failure (ALF) is a life-threatening disease caused by extensive hepatocyte dysfunction. Many treatments have been used to treat ALF, among which cell transplantation is the most advanced strategy. It can achieve the regeneration and repair of liver with the help of external cells, including stem cells, liver primary / progenitor cells, human induced pluripotent stem cell-derived hepatocytes (hiPSC-heps) and the like. Among these external cells, hiPSC-heps are considered as a promising cell source for ALF treatment due to their convenient induction strategy, excellent liver function and clear efficacy. Despite these advantages, hiPSC-heps injected intravenously are often attacked by host immune cells, leading to cell loss, functional impairment and non-targeted aggregation. In addition, dispersed hiPSC-heps infiltrate the liver, face challenges in cultivating the liver microenvironment conducive to culture, and are difficult to exert optimal therapeutic functions such as reducing damage, increasing proliferation and promoting liver regeneration. In addition, the ischemic, hypoxic or inflammatory conditions of the damaged liver can severely affect the survival ability of these transplanted hiPSC-heps. Therefore, there is still a need for new cell delivery strategies to improve the survival rate and function of hiPSC-heps in ALF treatment.

[0003] However, due to the lack of high precision and controllable preparation methods, conventional cell spheroids usually have the disadvantages of inaccurate size and uneven structure. Cell spheroids are prone to stick together during local implantation, leading to nutrient deficiency. Therefore, it is important to develop an effective strategy to achieve vascularized cell spheroids for hiPSC-heps to promote liver repair. SUMMARY

[0004] The purpose of the present invention is to solve the above technical problems, inspired by the vascularization of supporting cell structures in tissues and organs, we propose a simple microfluidic electrospray microcapsule technology to prepare biomimetic vascularized hiPSC-heps spheroids required for ALF treatment for in situ transplantation of liver repair, which is prepared by the method of microfluidic electrospray microcapsule technology, the method is simple, universal and easy to mass produce.

[0005] Technical solution: The bionic vascularized iPSC-hepatocyte spheroid of the application is a microcapsule with porous methacrylated hyaluronic acid (HAMA) as the shell and human induced pluripotent stem cell-derived hepatocyte (hiPSC-hep) spheroid as the inner phase, and the surface of the shell is adhered with human umbilical vein endothelial cells (HUVECs).

[0006] Further, the diameter of the inner phase of the microcapsule is 180-220 μm, and the pore size of the outer shell of the microcapsule is greater than 10 μm, which is sufficient for effective material exchange between cells.

[0007] Further, the bionic vascularized iPSC-hepatocyte spheroid is prepared by microfluidic electrospray microcapsule technology.

[0008] The preparation steps are as follows:

[0009] 1) Preparation of human induced pluripotent stem cell-derived hepatocytes (hiPSC-heps);

[0010] 2) The carboxymethyl cellulose (CMC) solution of hiPSC-heps is used as the inner phase, and the mixed solution of methacrylated hyaluronic acid (HAMA) and sodium alginate (Na-Alg) is used as the outer phase, which enters the inner channel and the outer channel of the microfluidic device respectively, and under the action of electrostatic force, the outlet of the microfluidic device is cut into droplets with core-shell structure;

[0011] 3) The droplets with core-shell structure prepared in step 2) are placed in a calcium chloride collection pool, the Na-Alg component reacts quickly with calcium ions to generate solid calcium alginate (Ca-Alg), and at the same time, the collection pool is irradiated with ultraviolet light, and the HAMA component is polymerized, so that the microcapsule with solidified Ca-Alg / HAMA shell can be obtained;

[0012] 4) The microcapsule obtained in step 3) is cultured, and when the hiPSC-heps form spheroids, the microcapsule is immersed in a sodium citrate aqueous solution, and then human umbilical vein endothelial cells (HUVECs) are adhered to the surface of the microcapsule shell, thereby obtaining a bionic vascularized iPSC-hepatocyte spheroid.

[0013] In step 2), the flow rate of the inner phase of the microfluidic device is 4 μL / min, the flow rate of the outer phase is 40 μL / min, the voltage is 6.5 kV, and the height is 4.5 cm. The mass concentration of the CMC solution of the inner phase hiPSC-heps is 1.0 wt%, and the outer phase contains 1.5 wt% of sodium alginate and 2.0 wt% of hyaluronic acid methacrylate aqueous solution.

[0014] In step 3), the calcium chloride collection pool is a 2.0 wt% CaCl2 aqueous solution.

[0015] Step 4) the mass concentration of the aqueous sodium citrate solution is 10.0 wt%.

[0016] The application also provides application of the prepared biomimetic vascularized iPSC-hepatic cell spheroids in preparation of a liver regeneration drug. Advantages

[0017] (1) The application designs a biomimetic vascularized iPSC-hepatic cell spheroid for liver regeneration, which has superior monodispersity, uniformity, stability and size controllability.

[0018] (2) The biomimetic vascularized iPSC-hepatic cell spheroid for liver regeneration is prepared by a microfluidic electrospray microcapsule technology, which has the advantages of simple method, convenient operation, strong repeatability, low technical requirement, strong universality, high flexibility and easy large-scale preparation.

[0019] (3) The biomimetic vascularized iPSC-hepatic cell spheroid for liver regeneration prepared by the application adheres HUVECs on the surface to form a vascularized network structure, promotes effective communication between hiPSC-heps and HUVECs, and provides barrier protection for the encapsulated hiPSC-hep spheroids to avoid attack by the immune system. Compared with dispersed cells, the cell-cell interaction in the cell spheroids improves the intercellular communication, helps to simulate the physiological heterogeneity structure and cell microenvironment of the liver, and can more effectively repair an acute liver failure. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the preparation process of the biomimetic vascularized iPSC-hepatic cell spheroid; Figure a is a schematic diagram of the microfluidic preparation and culture process of the biomimetic vascularized microcapsule; and Figure b is a schematic diagram of assembling and implanting the microcapsule into an ALF rat to perform liver regeneration.

[0021] Figure 2 is a schematic diagram of the preparation and regulation of the biomimetic vascularized iPSC-hepatic cell spheroid; Figure a is a schematic diagram of the microfluidic preparation process of the microcapsule; Figure b is a structure of the microfluidic microcapsule observed by a stereomicroscope; Figure c is a scanning electron microscope image of the entire microcapsule; Figures d and e are optical microscope images of the microcapsule with different Fouter / Finner ratios, and the scales are 200 μm; and Figure f is the overall size and core size distribution of the CMC / Ca-Alg-HAMA microcapsule under different parameters.

[0022] Figure 3 Schematic diagram of characterization of the bioengineered vascularized iPSC-hep spheroids: a, SEM image of the microcapsules encapsulating hiPSC-hep spheroids, scale bar 50 μm; b, confocal laser scanning image of hiPSC-hep spheroidal microcapsules stained with albumin / proliferating cell nuclear antigen (ALB / PCNA), scale bar 50 μm; c, schematic diagram of the selective degradation of the microcapsules and the coating process of HUVECs; d, laser scanning confocal microscope (LSCM) image of the communication between hiPSC-heps and HUVECs, scale bar 50 μm; e, SEM images of the interior (i) and surface (ii) of the microcapsules, scale bar 50 μm; f, viability of hiPSC-hep spheroids encapsulated in three different ratios of hydrogel shells.

[0023] Figure 4 Schematic diagram of the vascular network of the bioengineered vascularized iPSC-hep spheroids: a, schematic diagram of the bioengineered vascularized iPSC-hep spheroids encapsulated with hiPSC-hep spheroids and coated with HUVECs; b, confocal laser scanning image of a single microcapsule encapsulating hiPSC-hep spheroids (green) and HUVECs (red), scale bar 50 μm; c, schematic diagram of the assembly of the bioengineered vascularized iPSC-hep spheroids through the tight junctions of HUVECs, scale bar 100 μm; d and e, schematic diagrams of the assembly of the bioengineered vascularized iPSC-hep spheroids in a single row (d) and a sheet (e), scale bar 100 μm.

[0024] Figure 5 Schematic diagram of in vivo study to evaluate the neovascularization and targeted delivery capacity: a, schematic diagram of ALF rats receiving treatment of assembled bioengineered vascularized iPSC-hep spheroids; b-d, confocal laser scanning images of the vascular network, mCheery identifies HUVECs (red), GFP identifies hiPS-heps (green), DAPI stains the cell nucleus (blue). Scale bar 50 μm on day 1, 3, 7; e, schematic diagram of the characterization by confocal laser scanning, endothelial cells are represented by CD31, red label highlights HUVECs and rat endothelial cells, green label precisely locates HUVECs, scale bar 50 μm.

[0025] Figure 6 Schematic diagram of the in vivo therapeutic effect of the bioengineered vascularized iPSC-hep spheroids on the ALF rat model: a-c, determination of the ALT, AST, INR levels of the ALF rats in each group; d, detection of the location and intensity of DIR-stained hiPSC-heps in the ALF rats by a small animal imaging system; e, HE, Ki-67 staining images of each group, scale bar 50 μm; f, proportion of Ki-67 staining positive in the three groups. DETAILED DESCRIPTION

[0026] In order to deepen the understanding of the present application, the present application will be further described below in conjunction with examples and drawings, which are only used to explain the present application and do not constitute a limitation on the protection scope of the present application.

[0027] The human induced pluripotent stem cells (hiPSC) used in the following examples were purchased from the Chinese Academy of Sciences Stem Cell Bank, and HUVECs were purchased from the ATCC Cell Bank.

[0028] Example 1 Preparation method of biomimetic vascularized iPSC-hepatocyte spheroids

[0029] Preparation of human induced pluripotent stem cell-derived hepatocytes (hiPSC-heps) (Preparation steps cited prior art literature Chen, Sitong et al. Hepatic spheroids derived from human induced pluripotent stem cells in bio-artificial liver rescue porcine acute liver failure. Cell Research, (2019) 0:1-3):

[0030] Human induced pluripotent stem cells (hiPSC) were cultured in 6-well plates (5x105 cells per well) at 37°C, 5% CO2 using mTeSR1 medium containing Matrigel. Subsequently, the cells were cultured in RPMI1640 medium containing Activin a, BMP4, bFGF, B27 and Wnt3a for 1 day, and then transferred to RPMI1640 medium containing Activin a, BMP4 and bFGF for 3 days to stimulate the development of the final endoderm cells. To promote the formation of hepatoblasts, the endoderm cells were cultured in RPMI1640 medium containing KGF, SB431542 and B27 for 2 days, and then in RPMI1640 medium containing KGF, BMP4, BMP2, bFGF and B27 for 3 days. To promote the differentiation of hepatocytes into hepatic progenitor cells (HPCs), the hepatoblasts were cultured in DMEM / F12 medium containing B27, forskolin, SB431542 EGF, CHIR99021, LPA, Dex and S1P for 6-8 days. To produce mature hepatocytes (hiPSC-Heps), the HPCs were cultured in Williams'E medium containing B27, forskolin and SB431542 for 21 days.

[0031] (2) Preparation of biomimetic vascularized iPSC-hepatocyte spheroids

[0032] As shown in FIG. 2a, about 107hiPSC-heps prepared in step (1) were dissolved in 1 ml of carboxymethyl cellulose (CMC) solution. The 1.0 wt% CMC solution containing hiPSC-heps was used as the inner phase, and 5 ml of 1.5 wt% sodium alginate and 2.0 wt% hyaluronic acid methacrylate (HAMA) aqueous solution was used as the outer phase. The collection pool was 2.0 wt% CaCl2solution. The inner and outer solutions were coaxially flowed, so that the outer phase was wrapped around the inner phase. At the same time, the electrostatic effect of the electrospinning jet cut the flow into droplets, which were then collected in calcium chloride. After 1 min of ultraviolet irradiation, the HAMA component was polymerized, and microcapsules with solidified Ca-Alg / HAMA shells were obtained (FIG. 2b). The core-shell structure of the single CMC / Ca-Alg-HAMA microcapsule was confirmed by scanning electron microscopy (SEM) (FIG. 2c). Because of the excellent operability of the microfluidic electrospinning technology, we studied the correlation between the flow rates of the inner (Finner) and outer (Fouter) phases and the diameter of the core-shell microcapsule (FIG. 2d). It can be found that when the inner phase increases, the core layer diameter increases, and the shell thickness decreases (FIG. 2d (i, ii), 2f). Conversely, as the outer phase increases, the microcapsule diameter and the outer shell thickness increase, and the core layer diameter decreases (FIG. 2e (i, ii), 2g). In addition, we found that the diameter of the microcapsule decreases with the increase of the voltage (FIG. 2h). By dynamically adjusting the flow rate, collection distance and voltage, we finally determined that Finner was 4 μL / min, Fouter was 40 μL / min, the voltage was 6.5 kV, and the height was 4.5 cm. The resulting microspheres had excellent monodispersity (FIG. 2i). Under this condition, the inner phase diameter of the microcapsule was about 200 μm, and each microcapsule could encapsulate about 500 cells.

[0033] After 7 days of culture in a 37°C, 5% CO2 cell incubator, hiPSC-heps formed spheroids. Then the microcapsules were immersed in a 10.0 wt% sodium citrate solution for 2 hours, and the sodium citrate solution could selectively degrade Ca-Alg. Subsequently, HUVECs were adhered to the surface of the HAMA shell, and about 5 million HUVECs were cultured in each well to cover the surface of the hiPSC-hep spheroidal microcapsule, i.e. to obtain a biomimetic vascularized iPSC-liver cell spheroid (FIG. 1a).

[0034] The biomimetic vascularized iPSC-liver cell spheroid not only promotes communication between HUVECs and hiPSC-heps. Moreover, the surrounding network of HUVECs provides a protective barrier for the hiPSC-hep spheroid to avoid attack by immune cells and support nutrition. In addition, it can also improve the efficiency of vascular system regeneration. Therefore, this vascularized hiPSC-hep spheroid can more effectively repair an acutely failing liver (FIG. 1b).

[0035] Example 2 Characterization of the bioengineered vascularized iPSC-hepatocyte spheroids

[0036] The bioengineered vascularized iPSC-hepatocyte spheroids prepared in Example 1 were characterized. Scanning electron microscopy (SEM) showed that hepatocytes were tightly connected to each other and aggregated in the core of the nuclear shell to form spheroids (Figure 3a). The expression of liver functions was also evaluated by assessing the expression of albumin and the ability of cells to proliferate (Figure 3b). Due to the limited space between Ca-Alg hydrogels, the structure of HAMA hydrogels was relatively loose with large gaps, which was not conducive to the connection between cells. Then, we selectively degraded Ca-Alg using sodium citrate, while the HAMA hydrogel maintained the shell structure of the microcapsule, and subsequently HUVECs were adhered to the surface of the HAMA shell, as shown in Figure 3c. hiPSC-heps were observed to penetrate the shell and spatially communicate with HUVECs by using laser scanning confocal microscopy (LSCM). The SEM results further confirmed this LSCM phenomenon. On the shell of the hydrogel, not only did HUVECs adhere tightly to the outer layer (Figure 3e (i)), but also a large number of hiPSC-heps adhered firmly to the inner layer of the shell (Figure 3e (ii)). The effect of different component shell hydrogels on the function of hiPSC-heps spheroids was detected by cell counting kit 8 (CCK8) activity experiments. The results showed that the shell hydrogel with degraded Ca-Alg and HAMA hydrogel could better maintain the activity of hiPSC-heps spheroids compared to the Ca-Alg shell and Ca-Alg-HAMA shell (Figure 3f).

[0037] Example 3 Characterization of the bioengineered vascularized iPSC-hepatocyte spheroids vascular network

[0038] As shown in Figure 4a, the vascularized core-shell microspheres loaded with hiPSC-heps spheroids were successfully prepared. By LSCM observation, it was found that the hiPSC-heps and HUVECs were located between the inner and outer parts of the hydrogel shell, with a clear boundary (Figure 4b). The presence of hiPSC-heps spheroids enhanced the tight junctions of HUVECs. These microcapsules were staggered and formed a hexagonal close-packed tight connection on the plane (Figure 4c (i-iii)). Through three-dimensional reconstruction, each microcapsule presented a complete spherical shape, and the shell of HUVECs did not collapse (Figure 4c (iv)). Subsequently, we found that if multiple microcapsules were arranged in a straight line, after a period of culture, the HUVECs in contact between the two adjacent microcapsules would adhere to each other, forming a single-row bead structure, and the microcapsules were not easy to disperse (Figure 4d). Therefore, we further assembled these microcapsules into a sheet-shaped tissue. We found that the hexagonal close-packed microcapsules were arranged uniformly, forming a good plane, and the marker was tightly connected and not easy to disperse (Figure 4e).

[0039] Example 4 In vivo study of the targeted delivery ability of the biomimetic vascularized iPSC-hepatocyte spheroids

[0040] To demonstrate that the biomimetic vascularized iPSC-hepatocyte spheroids can be used to treat liver diseases. An ALF rat model was established by intraperitoneal injection of D-galactosamine (D-Gal). Then the damaged liver was treated with biomimetic vascularized iPSC-hepatocyte spheroids (Figure 5a). By making a 1 cm incision on the upper abdomen of the rat to expose the liver, multiple biomimetic vascularized iPSC-hepatocyte spheroid transplants were performed under the liver capsule. On the first day after transplantation, the hiPSC-heps spheroids were completely wrapped by the ring structure formed by vascular endothelial cells (Figure 5b). Over time, the hiPSC-heps gradually spread and showed a tendency to penetrate the HUVECs network. By the 7th day, the network structure of HUVECs remained intact, with tight connections between cells, but the internal hiPSC-heps had penetrated and dispersed throughout the liver (Figures 5c-d). In addition, by using specific anti-human CD31 antibodies (green fluorescently labeled HUVECs on transplanted microcarriers) and anti-human and anti-rat CD31 antibodies (red fluorescently labeled HUVECs on transplanted microcapsules and rat endothelial cells), we found that the green fluorescence overlapped with the red fluorescence, and the areas that did not overlap indicated the formation of a vascular network (Figure 5e).

[0041] Example 5 In vivo therapeutic effect of biomimetic vascularized iPSC-hepatocyte spheroids on ALF rat models

[0042] To further understand the efficacy of the biomimetic vascularized iPSC-heps spheroids in treating ALF, the liver function, coagulation, blood ammonia and other indicators of each group of mice were detected. The detection indexes include alanine aminotransferase (ALT) for evaluating the degree of liver cell damage, aspartate aminotransferase (AST) for evaluating the degree of liver cell damage, international normalized ratio (INR) for measuring the degree of coagulation function damage, and blood ammonia (NH3) for judging whether the liver metabolism is abnormal.

[0043] Four groups were set up in the experiment, including

[0044] Normal group - normal rats;

[0045] ALF group - rats induced by acute liver failure;

[0046] Spheroids group - rats induced by acute liver failure, treated with simple iPSC-heps cell spheroids;

[0047] Microcapsules group - rats induced by acute liver failure, treated with biomimetic vascularized iPSC-heps spheroids prepared by the present application.

[0048] The results show that the above indexes and survival rate of the liver transplanted rats in the Spheroids group and the Microcapsules group are significantly better than those in the ALF group (Fig. 6a-c). To further study the colonization of the microcapsules (biomimetic vascularized iPSC-liver cell spheroids) in the liver, small animal in vivo imaging was performed after transplantation, as shown in Fig. 6d. The results show that the transplanted microcapsules can concentrate in the liver area and exhibit considerable fluorescence intensity, without being transferred to other parts of the body. In addition, the microcapsules can maintain an ideal fluorescence intensity for up to 7 days. We also observed the pathological changes of rat liver tissue by hematoxylin-eosin (HE) staining. The main pathological changes in the ALF group were central and peripheral venous hepatocyte cystic necrosis, with a total of 6 points. The Spheroids group and the Microcapsules group were characterized by central vein hyperemia and hepatocyte vacuolization, with 2 points each. However, compared with the Microcapsules group, the degree of hyperemia and vacuolization in the Spheroids group was slightly heavier (Fig. 6e). In addition, the degree of cell proliferation was detected by Ki-67 staining method. Compared with the other two groups, the liver cell proliferation in the Microcapsules group was significantly improved (Fig. 6f). These results show that the biomimetic vascularized iPSC-liver cell spheroids can effectively promote liver repair.

[0049] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A bioengineered vascularized iPSC-hepatocyte spheroid, characterized in that, The biomimetic vascularized iPSC-liver spheroid is a microcapsule with a porous methacrylated hyaluronic acid shell and a human induced pluripotent stem cell-derived liver spheroid as the inner phase, and the surface of the shell is adhered with human umbilical vein endothelial cells.

2. The biologicallv vascularized iPSC-hepatocyte spheroid of claim 1, wherein, The diameter of the inner phase of the microcapsule is 180-220 μm, and the pore size of the outer shell of the microcapsule is greater than 10 μm.

3. The biologicallv vascularized iPSC-hepatocyte spheroid of claim 1, wherein, The biomimetic vascularized iPSC-liver spheroid is prepared by microfluidic electrospray microcapsule technology.

4. The biovicated iPSC-hepatocyte spheroid according to any one of claims 1-3, wherein, The preparation steps are as follows: 1) Preparation of human induced pluripotent stem cell-derived liver cells; 2) The carboxymethylcellulose solution of human induced pluripotent stem cell-derived liver cells is used as the inner phase, and the mixed solution of methacrylated hyaluronic acid and sodium alginate is used as the outer phase, which enters the inner channel and the outer channel of the microfluidic device respectively, and under the action of electrostatic force, the outlet of the microfluidic device is cut into droplets with core-shell structure; 3) The droplets with core-shell structure prepared in step 2) are placed in a calcium chloride collection pool, and the collection pool is irradiated with ultraviolet light to obtain microcapsules with solidified shells; 4) The microcapsules obtained in step 3) are cultured, and when the human induced pluripotent stem cell-derived liver cells form spheroids, the microcapsules are immersed in a sodium citrate aqueous solution, and then human umbilical vein endothelial cells are adhered to the surface of the microcapsule shell to obtain a biomimetic vascularized iPSC-liver spheroid.

5. The biologicallv vascularized iPSC-hepatocyte spheroid of claim 3, wherein, In step 2), the inner phase flow rate of the microfluidic device is 4 μL / min, the outer phase flow rate is 40 μL / min, the voltage is 6.5 kV, and the height is 4.5 cm.

6. The biologicallv vascularized iPSC-hepatocyte spheroid of claim 3, wherein, In step 2), the mass concentration of the inner phase human induced pluripotent stem cell-derived liver cells is 1.0 wt%, and the outer phase contains 1.5 wt% sodium alginate and 2.0 wt% methacrylated hyaluronic acid aqueous solution.

7. The biologicallv vascularized iPSC-hepatocyte spheroid of claim 3, wherein, In step 3), the calcium chloride collection pool is a 2.0 wt% CaCl2 aqueous solution.

8. The biologicallv vascularized iPSC-hepatocyte spheroid of claim 3, wherein, In step 4), the mass concentration of the sodium citrate aqueous solution is 10.0 wt%.

9. Use of the biomimetic vascularized iPSC-liver spheroid of any one of claims 1-8 in the preparation of a drug for liver regeneration.

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