A biomimetic vascularized iPSC-hepatocyte spheroid for liver regeneration and a preparation method thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a biomimetic vascularized iPSC-hepatocyte sphere for liver regeneration and a preparation method thereof. Background Art

[0002] Acute liver failure (ALF) is a serious life-threatening disease caused by extensive hepatocyte dysfunction. Many therapeutic approaches have been used to treat ALF, among which cell transplantation is the most advanced strategy. It can achieve liver regeneration and repair with the help of external cells, including stem cells, liver primary / progenitor cells, human induced pluripotent stem cell-derived hepatocytes (hiPSC-heps), etc. Among these external cells, hiPSC-heps are considered to be 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, resulting in their cell loss, functional impairment and non-targeted aggregation. In addition, dispersed hiPSC-heps infiltrate the liver and face challenges in cultivating a favorable liver microenvironment, making it difficult to exert optimal therapeutic functions, such as alleviating damage, increasing proliferation and promoting liver regeneration. In addition, ischemic, hypoxic or inflammatory conditions in the damaged liver can seriously affect the viability of these transplanted hiPSC-heps. Therefore, new cell delivery strategies are still needed 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, traditional cell spheroids usually have disadvantages such as inaccurate size and uneven structure. Cell spheroids tend to stick together during local implantation, resulting in nutrient deficiency. Therefore, developing an effective strategy to achieve vascularized cell spheroids is important for hiPSC-hepsin to promote liver repair. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to solve the above-mentioned technical problems. Inspired by the vascularity of supporting cell structures in tissues and organs, we proposed a simple microfluidic electrospray microencapsulation technology to prepare biomimetic vascularized hiPSC-heps spheres required for ALF treatment for in situ transplantation liver repair. The spheres are prepared by microfluidic electrospray microencapsulation technology, which is simple, versatile and easy to mass produce.

[0005] Technical solution: The bionic vascularized iPSC-hepatocyte spheroid described in the present invention is a microcapsule with porous methacryloyl hyaluronic acid (HAMA) as the outer shell and human induced pluripotent stem cell-derived hepatocyte (hiPSC-heps) spheroid as the inner phase, and human umbilical vein endothelial cells (HUVECs) are adhered to the surface of the outer shell.

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

[0007] Furthermore, the bionic vascularized iPSC-hepatocyte spheroids are prepared by using microfluidic electrospray microencapsulation 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 was used as the inner phase, and the mixed solution of methacryloyl hyaluronic acid (HAMA) and sodium alginate (Na-Alg) was used as the outer phase, which entered the inner channel and outer channel of the microfluidic device respectively. Under the action of electrostatic force, the outlet of the microfluidic device was cut into droplets with a core-shell structure;

[0011] 3) placing the droplets with a core-shell structure prepared in step 2) into a calcium chloride collection pool, wherein the Na-Alg component reacts rapidly with calcium ions to generate solid calcium alginate (Ca-Alg), and simultaneously irradiating the collection pool with ultraviolet light to polymerize the HAMA component, thereby obtaining a microcapsule with a solidified Ca-Alg / HAMA shell;

[0012] 4) Cultivating the microcapsules obtained in step 3) until the hiPSC-hepsin forms spheroids, immersing the microcapsules in a sodium citrate aqueous solution, and then adhering human umbilical vein endothelial cells (HUVECs) to the surface of the microcapsule shell to obtain biomimetic vascularized iPSC-hepatocyte spheroids.

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

[0014] The calcium chloride collection pool described in step 3) is a CaCl2 aqueous solution with a mass concentration of 2.0 wt%.

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

[0016] The present invention also provides the use of the bionic vascularized iPSC-hepatocyte spheroid prepared above in the preparation of a drug for liver regeneration. Beneficial Effects

[0017] (1) The present invention designs a biomimetic vascularized iPSC-hepatocyte spheroid for liver regeneration, which has excellent monodispersity, uniformity, stability and size controllability.

[0018] (2) The bionic vascularized iPSC-hepatocyte spheroids for liver regeneration provided by the present invention are prepared by microfluidic electrospray microencapsulation technology, which is simple, easy to operate, highly repeatable, has low technical requirements, strong versatility, high flexibility, and is easy to prepare on a large scale.

[0019] (3) The bionic vascularized iPSC-hepatocyte spheroids for liver regeneration prepared by the present invention adhere to HUVECs on their surface to form a vascularized network structure, which 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-to-cell interactions within the cell spheroids improve cell-to-cell communication, help simulate the physiological heterogeneous structure and cellular microenvironment of the liver, and can more effectively repair acutely failing livers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the preparation process of bionic vascularized iPSC-hepatocyte spheroids: Figure a is a schematic diagram of the microfluidic preparation and culture process of bionic vascularized microcapsules; Figure b is a schematic diagram of the microcapsule assembly and implantation into ALF rats for liver regeneration.

[0021] Figure 2 Schematic diagram of the preparation and regulation of bionic vascularized iPSC-hepatocyte spheroids: Figure a is a schematic diagram of the microfluidic preparation process of microcapsules; Figure b is the observation of the microfluidic microcapsule structure using a stereo microscope; Figure c is a scanning electron microscope image of the entire microcapsule; Figures d and e are optical microscope images of Fouter / Finner microcapsules of different scales, with a scale of 200 μm respectively; Figures f, g, h, and i are the overall size and core size distribution of CMC / Ca-Alg-HAMA microcapsules under different parameters.

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

[0023] Figure 4 Schematic diagram of the vascular network of bionic vascularized iPSC-hepatocyte spheroids: Figure a is a schematic diagram of bionic vascularized iPSC-hepatocyte spheroids encapsulated with hiPSC-hep spheroids and coated with HUVECs; Figure b is a confocal laser scanning image of a single microcapsule, encapsulating hiPSChep spheroids (green) and HUVECs (red), with a scale bar of 50 μm; Figure c is a schematic diagram of the assembly of bionic vascularized iPSC-hepatocyte spheroids tightly connected by HUVECs, with a scale bar of 100 μm; Figures d and e are schematic diagrams of the single row (d) and sheet (e) assembly of bionic vascularized iPSC-hepatocyte spheroids, with a scale bar of 100 μm.

[0024] Figure 5 Schematic diagram of in vivo studies to evaluate angiogenesis and targeted delivery capabilities: Figure a is a schematic diagram of ALF rats receiving assembled biomimetic vascularized iPSC-hepatocyte spheroids for treatment; Figures b and d are schematic diagrams of confocal laser scanning vascular networks, mCheery identifies HUVECs (red), GFP identifies hiPS-heps (green), and DAPI stains cell nuclei (blue). The scale bar represents 50 μm on days 1, 3, and 7; Figure e is a schematic diagram of characterization using confocal laser scanning, with endothelial cells represented by CD31, red markers highlighting HUVECs and rat endothelial cells, and green markers accurately locating HUVECs, and the scale bar corresponds to 50 μm.

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

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

[0027] The human induced pluripotent stem cells (hiPSCs) used in the following examples were purchased from the Stem Cell Bank of the Chinese Academy of Sciences, 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) (the preparation steps are cited in the prior art document Chen, Sitong et al. Hepatic spheroids derived from human induced pluripotentstem cells in bio-artificial liver rescue porcine acute liver failure. Cell Research, (2019) 0: 1-3):

[0030] Human induced pluripotent stem cells (hiPSCs) were cultured in 6-well plates (5 × 105 cells per well) using mTeSR1 medium containing Matrigel at 37°C and 5% CO2. 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 final endoderm cells. To promote the formation of hepatoblasts, endoderm cells were cultured in RPMI1640 medium containing KGF, SB431542, and B27 for 2 days, and then cultured in RPMI1640 medium containing KGF, BMP4, BMP2, bFGF, and B27 for 3 days. To promote the differentiation of hepatocytes into hepatic progenitor cells (HPCs), hepatoblasts were cultured in DMEM / F12 medium containing B27, forskolin, SB431542 EGF, CHIR99021, LPA, Dex, and S1P for 6 to 8 days. To generate mature hepatocytes (hiPSC-Heps), 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] like Figure 2 As shown in a, hiPSC-heps cells prepared in about 107 steps (1) are dissolved in 1 ml of carboxymethyl cellulose (CMC) solution, with 1.0 wt% CMC solution containing hiPSC-heps as the inner phase, 5 ml of an aqueous solution of 1.5 wt% sodium alginate and 2.0 wt% hyaluronic acid methacryloyl (HAMA) as the outer phase, and a collection pool of 2.0 wt% CaCl2 solution. The inner and outer solutions flow coaxially, so that the outer phase wraps around the inner phase. At the same time, the electrostatic effect of the electrospray cuts the flow into droplets, and then the droplets are collected in calcium chloride and irradiated with ultraviolet light for 1 min to polymerize the HAMA component, thereby obtaining a solidified Ca-Alg / HAMA shell microcapsule ( Figure 2 b), The core-shell structure of single CMC / Ca-Alg-HAMA microcapsules was confirmed by scanning electron microscopy (SEM). Figure 2 c). Due to the excellent operability of microfluidic electrospray technology, we studied the correlation between the flow rate of the inner (Finner) phase and the outer (Fouter) phase and the diameter of the core-shell microcapsules ( Figure 2 d). It can be found that when the inner phase increases, the core layer diameter increases, while the shell thickness decreases ( Figure 2 d (i, ii), 2f). On the contrary, with the increase of the external phase, the microcapsule diameter and shell thickness increased, and the core layer diameter decreased ( Figure 2 e (i, ii), 2g). In addition, we found that the diameter of the microcapsules decreased with increasing voltage ( Figure 2 h). By dynamically adjusting the flow rate, collection distance and voltage, we finally determined that the Finner was 4 μL / min, the Fouter was 40 μL / min, the voltage was 6.5 kV, and the height was 4.5 cm. The obtained microspheres had excellent monodispersity ( Figure 2 i) Under this condition, the inner phase diameter of the obtained microcapsules is about 200 μm, and each microcapsule can encapsulate about 500 cells.

[0033] After 7 days of culture in a 37°C, 5% CO2 cell culture incubator, hiPSC-heps formed spheroids. The microcapsules were then immersed in a 10.0 wt% sodium citrate solution for 2 hours, which selectively degraded Ca-Alg. HUVECs were then 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 spherical microcapsules, thus obtaining biomimetic vascularized iPSC-hepatocyte spheroids ( Figure 1 a).

[0034] The biomimetic vascularized iPSC-hepatocyte spheroids can not only promote the communication between HUVECs and hiPSC-heps. And the surrounding HUVECs network provides a protective barrier for the hiPSC-hep spheres to avoid the attack of immune cells and support nutrition. In addition, it can also improve the efficiency of vascular system regeneration. Therefore, this vascularized hiPSC-hep sphere can more effectively repair the acute failure liver, ( Figure 1 b).

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

[0036] The biomimetic vascularized iPSC-hepatocyte spheroids prepared in Example 1 were characterized. Scanning electron microscopy (SEM) showed that the hepatocytes were aggregated in the center of the core shell and were closely connected to each other to form a spheroid ( Figure 3 a). Albumin expression and cell proliferation capacity were also evaluated for the effects on liver function expression levels ( Figure 3 b) Since the gaps between the Ca-Alg hydrogels are limited, the structure of the HAMA hydrogel is relatively non-dense and has large gaps, which is not conducive to the connection between cells. Then, we used sodium citrate to selectively degrade Ca-Alg, while the HAMA hydrogel maintained the shell structure of the microcapsule, and then HUVECs were attached to the surface of the HAMA shell, as shown in Figure 3 c. Laser scanning confocal microscopy (LSCM) was used to observe that hiPSC-heps were able to penetrate the shell and communicate with HUVECs in space. SEM results further confirmed this LSCM phenomenon. On the shell of the hydrogel, not only did HUVECs adhere tightly to the outer layer ( Figure 3 e (i)), and a large number of hiPSC-hepsin also firmly adhered to the inner layer of the shell ( Figure 3 e (ii)). The effects of shell hydrogels with different compositions on the function of hiPSC-heps spheroids were detected by cell counting kit 8 (CCK8) activity assay. The results showed that compared with Ca-Alg shell and Ca-Alg-HAMA shell, HAMA hydrogel shell with degraded Ca-Alg could better maintain the activity of hiPSC-heps spheroids ( Figure 3 f).

[0037] Example 3 Schematic diagram of the characterization of the vascular network of biomimetic vascularized iPSC-hepatocyte spheroids

[0038] like Figure 4 As shown in a, vascularized core-shell microspheres loaded with hiPSC-heps spheres were successfully prepared. LSCM observation revealed that hiPSC-heps and HUVECs were located between the inner and outer parts of the hydrogel shell, and the boundary was clearly visible ( Figure 4b). The presence of hiPSC-heps spheres enhances the close communication of HUVECs. These microcapsules are staggered and form hexagonal tight connections on the plane ( Figure 4 c (i-iii)). Through three-dimensional reconstruction, each microcapsule presents a complete spherical shape, and the shell of HUVECs has not collapsed ( Figure 4 c (iv)). Subsequently, we found that if multiple microcapsules were arranged in a straight line, after a period of culture, the HUVECs in contact with two adjacent microcapsules would adhere to each other to form a single-row beaded structure, and the microcapsules were not easy to disperse ( Figure 4 d). Therefore, we further assembled these microcapsules into sheet-like tissues. We found that the closely arranged hexagonal microcapsules were evenly arranged, forming a good plane, and the markers were tightly connected and not easy to disperse ( Figure 4 e).

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

[0040] To demonstrate that 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, biomimetic vascularized iPSC-hepatocyte spheroids were used to treat damaged livers ( Figure 5 a). A 1 cm incision was made in the rat's upper abdomen to expose the liver, and multiple biomimetic vascularized iPSC-hepatocyte spheroid transplantations were performed under the liver capsule. On the first day after transplantation, the hiPSC-heps spheroids were completely encapsulated by the ring-like structure formed by vascular endothelial cells ( Figure 5 b). Over time, hiPSC-heps gradually spread and showed a tendency to penetrate the HUVECs network. By day 7, the HUVEC network structure remained intact and the cells were tightly connected, but the hiPSC-heps inside had infiltrated and dispersed throughout the liver ( Figure 5 cd). In addition, by using specific anti-human CD31 antibodies (green fluorescence labels HUVECs on transplanted microcarriers) and anti-human and anti-rat CD31 antibodies (red fluorescence labels HUVECs on transplanted microcapsules and rat endothelial cells), we found that green fluorescence overlapped with red fluorescence, and non-overlapping areas indicated the formation of vascular networks ( Figure 5 e).

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

[0042] In order to further understand the efficacy of biomimetic vascularized iPSC-heps spheres in the treatment of ALF, the liver function, coagulation, blood ammonia and other indicators of each group of mice were tested. The test indicators include alanine aminotransferase (ALT) to evaluate the degree of liver cell damage, aspartate aminotransferase (AST) to evaluate the degree of liver cell damage, international normalized ratio (INR) to measure the degree of coagulation damage, and blood ammonia (NH3) to determine whether the liver metabolism is abnormal.

[0043] The experiment set up four groups, including

[0044] Normal group—normal rats;

[0045] ALF group—rats induced with acute liver failure;

[0046] Spheroids group - rats induced with acute liver failure, treated with simple iPSC-hepsin cell spheres;

[0047] Microcapsules group: rats induced with acute liver failure, treated with the biomimetic vascularized iPSC-heps spheres prepared in Example 1 of the present invention.

[0048] The results showed that the above indicators and survival rates of liver transplanted rats in the Spheroids group and Microcapsules group were significantly better than those in the ALF group ( Figure 6 ac). To further investigate the colonization of microcapsules (biomimetic vascularized iPSC-hepatocyte spheroids) in the liver, in vivo imaging of small animals was performed after transplantation, e.g. Figure 6 d. The results showed that the transplanted microcapsules could be concentrated in the liver area and exhibit considerable fluorescence intensity without being transferred to other parts of the body. In addition, the microcapsules could maintain the ideal fluorescence intensity for up to 7 days. We also used hematoxylin-eosin (HE) staining to observe the pathological changes in rat liver tissue. The main pathological changes in the ALF group were cystic necrosis of hepatocytes in the pericentral veins, with a total of 6 points. Both the Spheroids group and the Microcapsules group were characterized by congestion of the central veins and vacuolation of hepatocytes, with 2 points each. However, compared with the Microcapsules group, the degree of congestion and vacuolation in the Spheroids group was slightly more severe ( Figure 6 e). In addition, Ki-67 staining was used to detect the degree of cell proliferation. Compared with the other two groups, the proliferation of hepatocytes in the Microcapsules group was significantly improved ( Figure 6 f). These results suggest that biomimetic vascularized iPSC-hepatocyte spheroids can effectively promote liver repair.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic vascularized iPSC-hepatocyte spheroid, characterized in that: The preparation steps are as follows: 1) Preparation of human induced pluripotent stem cell-derived hepatocytes; 2) A carboxymethyl cellulose solution of human induced pluripotent stem cell-derived hepatocytes was used as the inner phase, and a mixed solution of methacryloyl hyaluronic acid and sodium alginate was used as the outer phase, which entered the inner channel and outer channel of the microfluidic device respectively. Under the action of electrostatic force, the outlet of the microfluidic device was cut into droplets with a core-shell structure; the inner phase flow rate of the microfluidic device was 4 μL / min, the outer phase flow rate was 40 μL / min, the voltage was 6.5 kV, and the height was 4.5 cm; 3) placing the droplets with a core-shell structure prepared in step 2) into a calcium chloride collection pool, and irradiating the collection pool with ultraviolet light to obtain microcapsules with solidified shells; 4) Cultivating the microcapsules obtained in step 3) until the human induced pluripotent stem cell-derived hepatocytes form spheroids, immersing the microcapsules in a sodium citrate aqueous solution, and then adhering human umbilical vein endothelial cells to the surface of the microcapsule shell to obtain biomimetic vascularized iPSC-hepatocyte spheroids.

2. The preparation method according to claim 1, characterized in that: Step 2) The mass concentration of human induced pluripotent stem cell-derived hepatocytes in the inner phase is 1.0 wt%, and the outer phase contains sodium alginate with a mass concentration of 1.5 wt% and hyaluronic acid methacryloyl aqueous solution with a mass concentration of 2.0 wt%.

3. The preparation method according to claim 1, characterized in that: The calcium chloride collection pool described in step 3) is a CaCl2 aqueous solution with a mass concentration of 2.0 wt%.

4. The preparation method according to claim 1, characterized in that: The mass concentration of the sodium citrate aqueous solution in step 4) is 10.0 wt%.

5. The biomimetic vascularized iPSC-hepatocyte spheroid prepared by any one of the preparation methods of claims 1 to 4, characterized in that: The bionic vascularized iPSC-hepatocyte sphere is a microcapsule with porous methacryloyl hyaluronic acid as the outer shell and human induced pluripotent stem cell-derived hepatocyte sphere as the inner phase, and human umbilical vein endothelial cells are adhered to the surface of the outer shell.

6. The biomimetic vascularized iPSC-hepatocyte spheroid according to claim 5, characterized in that: The inner phase diameter of the microcapsule is 180-220 μm, and the pore size of the microcapsule shell is greater than 10 μm.

7. Use of the biomimetic vascularized iPSC-hepatocyte spheroid according to any one of claims 1 to 6 in the preparation of drugs for liver regeneration.

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