Method for enhancing cell therapy function by mechanical preconditioning and applications thereof

Abstract

The application discloses a method for enhancing cell treatment function by mechanical pretreatment and application, which is characterized in that the method is to culture and / or differentiate the cells to be treated in a biomimetic mechanical microenvironment, and the viscoelasticity of the microenvironment is matched with the target healthy liver. The application also provides a functional enhanced cell-hydrogel microsphere composite prepared by the method, a tissue patch comprising the same and application of the tissue patch in preparation of a medicine for treating liver diseases. The application adjusts the viscoelasticity parameters (such as stress relaxation half-life and loss tangent) of the microenvironment, adjusts the ROCK / F-actin / YAP mechanical signal pathway in the cells, and systematically enhances the functional maturity and stability of the cells. In an acute liver failure model, the hepatocyte-like cells obtained by pretreatment by the method can not only effectively supplement liver function, but also actively correct abnormal mechanical signals of the abnormal host tissues, so that the function supplementing and microenvironment repairing are realized in a synergistic treatment. The application provides a new general, controllable and efficient strategy for cell treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and regenerative medicine technology. Specifically, it relates to a strategy for enhancing the therapeutic function of cells by pretreating their mechanical microenvironment, a functionally enhanced cell-hydrogel microsphere complex prepared based on this strategy, a tissue patch containing the complex, and their application in the preparation of drugs for treating liver diseases (especially acute liver failure). Background Technology

[0002] Acute liver failure (ALF) is a life-threatening clinical syndrome characterized by a rapid loss of liver function far exceeding the liver's regenerative capacity. Currently, orthotopic liver transplantation is the gold standard for treating ALF, but its application is limited by a severe shortage of donor livers. Therefore, developing alternative cell therapy methods, such as transplantation of hepatocyte-like cells (HLCs) derived from stem cells, has become a research hotspot. Human adipose-derived stem cells (hADSCs) are considered ideal seed cells for generating HLCs due to their abundant availability, ease of acquisition, and multi-lineage differentiation potential.

[0003] However, existing in vitro stem cell differentiation protocols mainly rely on the addition of a series of growth factors and chemical inducers. The resulting HLCs often lack functional maturity and exhibit unstable cell function. After being removed from the induction environment or transplanted into the body, their function rapidly deteriorates, leading to poor therapeutic effects. Recent studies suggest that this insufficient efficacy may be related to the difficulty of transplanted cells adapting to the significantly altered pathological microenvironment in the diseased liver. Current techniques largely attempt to enhance cell tolerance to harsh environments by pretreating cells using biochemical methods (such as the use of antioxidants and anti-inflammatory drugs).

[0004] It is noteworthy that, in addition to biochemical signals, the mechanical microenvironment plays a crucial role in regulating cell behavior, maintaining tissue homeostasis, and disease progression. Under pathological conditions, the mechanical properties of tissues (such as stiffness and viscoelasticity) undergo significant alterations. For example, in liver fibrosis, cirrhosis, and even ALF, the liver's mechanosensory pathways are abnormally activated, transforming the tissue from a healthy state of rapid stress relaxation to a pathological state of slow stress relaxation and increased viscosity. Current technologies have failed to fully recognize and utilize the altered mechanical properties of diseased tissues to design and optimize cell therapy strategies. Therefore, there is an urgent need to develop a novel cell pretreatment method that can mimic the mechanical properties of target healthy tissues, fundamentally enhance cell functional maturity, and proactively correct the pathological microenvironment. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method and application for enhancing cell therapy function through mechanical pretreatment. Specifically, this invention aims to provide a novel strategy for systematically enhancing cell functional maturity, stability, and active repair capabilities against pathological microenvironments by pretreating cells in a biomimetic microenvironment that simulates the mechanical properties of target healthy tissue.

[0006] This invention, through systematic research, reveals for the first time a crucial shift in liver viscoelasticity during acute liver failure (ALF): compared to the rapid stress relaxation characteristics of healthy livers, the stress relaxation half-life (t1 / 2) of ALF livers is significantly prolonged, exhibiting slower stress dissipation and a more elastic, solid-like state. Simultaneously, mechanosensory pathways (such as RhoGTPase and Hippo / YAP) are abnormally activated in ALF livers. Based on these findings, this invention proposes an innovative "mechanical preconditioning" strategy.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for enhancing cell therapy function through mechanical pretreatment, the method comprising: The cells to be treated are cultured and / or differentiated in a biomimetic mechanical microenvironment whose viscoelastic properties match those of a target healthy liver.

[0008] In a preferred embodiment of the present invention, the target healthy tissue is the liver, and the biomimetic mechanical microenvironment is characterized by: under compression testing, its stress relaxation percentage at 60 seconds is 40% to 70%, and / or its stress relaxation half-life (t1 / 2) is 5 to 25 seconds; under shear rheological testing, its loss tangent (tanδ) is 0.2 to 0.6.

[0009] As a preferred embodiment of the present invention, the biomimetic mechanical microenvironment is provided by a tunable viscoelastic hydrogel material having an interpenetrating polymer network (IPN) structure.

[0010] As a preferred embodiment of the present invention, the interpenetrating polymer network hydrogel is composed of alginate (Alg) and decellularized extracellular matrix (dECM); the alginate solution is 3% (w / v) sodium alginate dissolved in serum-free DMEM, mixed with a Ca-EDTA complex and adjusted to pH 7.4; the dECM is prepared from porcine liver by freeze-thaw, decellularization, and pepsin digestion, with a concentration of 15 mg / mL.

[0011] As a preferred embodiment of the present invention, the hydrogel is in the form of microspheres with a diameter of 200 μm to 250 μm, and is prepared by microfluidic control: the aqueous phase is a mixture of Alg and dECM, the oil phase is 20% (v / v) Span80 cyclohexane, the two-phase flow ratio is 1:4, and it is crosslinked in a cyclohexane bath containing 1% acetic acid at 37°C for 30 min.

[0012] In a preferred embodiment of the present invention, the cells to be treated are human adipose-derived stem cells (hADSCs) with a cell loading density of 2 × 10⁻⁶. 6 cells / mL, co-cultured in a low-adsorption 24-well plate for 3 days.

[0013] As a preferred embodiment of the present invention, the method further includes inducing the cells to be treated (such as hADSCs) to differentiate into functionally enhanced hepatocyte-like cells (HLCs) using a liver differentiation induction medium in the biomimetic mechanical microenvironment.

[0014] In a preferred embodiment of the present invention, the differentiation induction process includes a pre-induction stage, a differentiation induction stage, and a maturation stage: Pre-induction (2 days): Low-glucose DMEM containing EGF 20 ng / mL and βFGF 10 ng / mL. Differentiation induction (7 days): low glucose DMEM containing HGF 20 ng / mL, βFGF 10 ng / mL, and NAM 0.61 mg / mL; Maturation (14 days): Low-sugar DMEM containing OSM 20ng / mL, dexamethasone 1μM, and 1×ITS; Inhibitors available include: ROCK inhibitor Y-27632 10μM and YAP inhibitor verteporfin 0.5μM.

[0015] Specifically, the pre-induction phase may use a culture medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (βFGF); the differentiation induction phase may use a culture medium containing hepatocyte growth factor (HGF), βFGF and nicotinamide (NAM); and the maturation phase may use a culture medium containing cytosolic antagonist M (OSM), dexamethasone and ITS supplement.

[0016] In a preferred embodiment of the present invention, the method modulates the activity of intracellular mechanotransmission pathways by controlling the viscoelasticity of the biomimetic mechanical microenvironment. These pathways include the ROCK (Rho-associated coiled-coil-containing protein kinase) / F-actin / YAP (Yes-associated protein) signaling axis. Preferably, by providing a rapid stress relaxation mechanical microenvironment matching that of a healthy liver, the activation of ROCK signaling and nuclear translocation of YAP are inhibited, thereby promoting functional maturation of the cell.

[0017] In a second aspect, the present invention provides a functionally enhanced cell-hydrogel microsphere complex prepared by the method described in the first aspect, comprising: (a) Hydrogel microspheres with tunable viscoelasticity as a biomimetic mechanical microenvironment; and (b) Cells loaded on or inside the surface of the hydrogel microspheres, wherein the functional maturity and stability of the cells are enhanced after the mechanical pretreatment.

[0018] Thirdly, the present invention provides a tissue patch for cell transplantation, comprising: (a) an adhesive underlayer for adhering the tissue patch to the surface of a target tissue; and (b) A functional layer comprising the cell-hydrogel microsphere complex described in the second aspect.

[0019] Preferably, the adhesive bottom layer is composed of dopamine-modified hyaluronic acid (HA-DA) hydrogel; HA-DA synthesis: 1% HA reacts with EDC / NHS (1:1) and dopamine (1:1), followed by lyophilization after 72 hours of dialyzing. The functional layer is a polyamino polymer-poly(acrylic acid-methacrylic acid-N-(methacryloylamino)succinimide) copolymer (PAMN) hydrogel patch; PAMN is prepared by polymerizing AA / MA / NHSMA and mixing it with ε-polylysine; the double-layer patch is molded by pressing at 60°C for 5 minutes.

[0020] Fourthly, the present invention provides the use of the method described in the first aspect, the cell-hydrogel microsphere complex described in the second aspect, or the tissue patch described in the third aspect in the preparation of a medicament for treating acute liver failure.

[0021] In a preferred embodiment of the present invention, the liver disease is acute liver failure (ALF).

[0022] As a preferred embodiment of the present invention, the application includes: transplanting the functionally enhanced cell-hydrogel microsphere complex or the tissue patch to the site of liver injury to supplement liver function, downregulate abnormally activated YAP signaling in the host liver, reduce oxidative stress levels, inhibit inflammatory responses, and promote liver tissue regeneration.

[0023] The present invention has the following beneficial effects: 1. Fundamentally Enhanced Cellular Functional Maturation: This invention overcomes the limitations of traditional biochemical factor-dependent induction methods by pretreating cells for the first time through precise simulation of the mechanical microenvironment (such as viscoelasticity) of the target healthy tissue. This "mechanical preconditioning" strategy can directly regulate intracellular mechanotransmission pathways such as ROCK / F-actin / YAP, systematically enhancing the expression of cell-specific functional markers (HNF4A, ALB, CYP3A4) at the transcriptional and protein levels. The resulting cells exhibit functional maturity and stability far exceeding those induced by existing technologies, and maintain a high level of function even after removal from the induction environment.

[0024] 2. Achieving a dual synergistic therapeutic effect of "cellular function replenishment" and "active correction of the pathological microenvironment": The cells prepared in this invention, because they are "calibrated" in a healthy mechanical environment, not only have enhanced functions, but also, after transplantation, can actively intervene in and partially reverse the abnormal mechanical signal state of the host diseased tissue through normal mechanical signal feedback and intercellular interactions (such as downregulating overactivated YAP signals). This invention thus achieves a synergistic effect of "treating the symptoms" (replenishing function) and "treating the root cause" (correcting the microenvironment), resulting in higher quality tissue regeneration and functional recovery; it can also reduce oxidative stress (MDA, O2). - It inhibits inflammation (Tnf, Nos2, Nlrp3) and upregulates antioxidant (Nqo1, Ho1, Gpx1) and anti-inflammatory (Il10, Arg1) genes.

[0025] 3. Providing a new paradigm for cell manufacturing with strong versatility and precise control: The mechanical pretreatment platform upon which this invention relies (such as Alg-dECMIPN hydrogel microspheres) has the characteristic of precise and independent controllability of mechanical parameters (such as stress relaxation time and loss modulus). This makes the method highly versatile and scalable. By simply adjusting the mechanical properties of the platform to the parameters of different target healthy livers, it can be used to pretreat corresponding stem cells, providing a new and engineerable technical path for stem cell-based regenerative medicine.

[0026] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Results of viscoelasticity assay of ALF-affected liver; Figure 2 Analysis of the mechanosensory pathway in the liver of patients with ALF; Figure 3 Preparation and mechanical characterization of dECM-Alg interpenetrating network hydrogel microspheres with customizable viscoelastic properties; Figure 4 Particle size distribution and microstructure characterization of hydrogel microspheres; Figure 5 Loading and activity detection of hADSCs on hydrogel microspheres; Figure 6 Results of hepatic differentiation of hADSCs in biomimetic microspheres; Figure 7 Immunofluorescence staining and quantitative analysis of HNF4α and ALB expression; Figure 8 Liver function characteristics of differentiated HLCs (CYP3A4 activity and secretory function); Figure 9 Characterization of the F-actin / ROCK / YAP signaling pathway in cells on microspheres with different mechanical properties; Figure 10 Schematic diagram of the regulatory role and mechanism of the ROCK / YAP signaling pathway in liver differentiation; Figure 11 Preparation and characterization of JanusHA-PAMN tissue patches; Figure 12 The therapeutic effect of biomimetic mechanical pretreated microspheres-HLCs on CCl4-induced ALF; Figure 13 Expression levels of liver function-related genes in liver tissue after treatment; Figure 14 Assessment of oxidative stress levels in liver tissue after treatment; Figure 15 Expression levels of inflammation-related genes in liver tissue after treatment; Figure 16 Immunofluorescence staining and quantitative analysis of pro-inflammatory markers NOS2 and TNFα in liver tissue sections; Figure 17 Changes in the mechanical properties of liver tissue and reversal of mechanosensory pathways after treatment; Figure 18 The therapeutic effect of biomimetic mechanical pretreated microspheres-HLCs on ALF induced by partial hepatectomy (PHx); Figure 19 Evaluation of the liver regeneration function of biomimetic mechanically pretreated microspheres-HLCs; Figure 20 qPCR analysis of hepatocyte-related genes after treatment; Figure 21 Assessment of oxidative stress levels in liver tissue after hepatectomy; Figure 22 Assessment of inflammation levels in liver tissue after hepatectomy; Figure 23 Long-term liver regeneration effect assessment; Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] In the description of this invention, it should be understood that the terms "upper," "middle," "outer," "inner," etc., which indicate orientation or positional relationship, are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the components or elements referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as limiting this invention.

[0031] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0032] Example 1: Identification of viscoelasticity and mechanosensory pathways in the liver of mice with acute liver failure (ALF) 1. Animal Models and Sample Preparation To investigate the changes in liver mechanobiological properties under ALF (alcoholic liver failure) conditions, this study used a carbon tetrachloride (CCl4)-induced mouse ALF model. All animal experiments were approved by the Animal Protection and Use Committee of Sun Yat-sen University (Approval No.: SYSU-IACUC-2026-000433). Six- to eight-week-old male C57BL / 6J mice were intraperitoneally injected with a 30% (volume ratio, dissolved in flaxseed oil) CCl4 solution at a dose of 5 μL / g body weight. Mice were sacrificed 24 hours after injection, and liver tissue was collected for subsequent analysis.

[0033] 2. Compression test Disc-shaped samples (2 mm thick, 8 mm in diameter) were obtained from mouse livers using a biopsy puncture device. The samples were uniaxially compressed at a constant deformation rate of 1 mm / min on a hydrogel biomechanical testing instrument until a strain of 10% was achieved. Young's modulus was calculated by linearly fitting the stress-strain data (from 5% to 10% strain). Furthermore, the stress relaxation response was quantified after applying and maintaining a 10% compressive strain.

[0034] 3. Rheological analysis Liver tissue was evaluated using a rotational rheometer at room temperature. The sample was placed in contact with the upper pressure plate under a minimum normal force of 0.01 N. Dynamic time scans were performed (constant strain 2%, oscillation frequency 1 rad / s), followed by stress relaxation tests induced by instantaneous application of 10% strain.

[0035] 4. RNA sequencing and bioinformatics analysis Total RNA was extracted from liver tissues of healthy controls and CCl4-induced ALF model groups (n=3 per group), and transcriptome sequencing was performed using the Illumina platform. Sequencing data were processed and analyzed on the OmicStudio and Sangerbox online platforms.

[0036] 5. Results Experimental results are as follows Figure 1 As shown. Compared to a healthy liver ( Figure 1 Compared to A), the livers of ALF mice were grayish-white, and the tissue texture was significantly altered. Figure 1 B). Compression test ( Figure 1 C) stress relaxation curve ( Figure 1 D) and quantitative analysis ( Figure 1 E and 1F showed that healthy livers had a shorter stress relaxation half-life (t1 / 2) and lower residual stress at 60 seconds; while ALF livers had a significantly prolonged t1 / 2 and higher residual stress, suggesting a slower stress relaxation process. Rheological analysis ( Figure 1 G-1J confirmed that the liver's internal structural remodeling capacity and external stress dissipation capacity were significantly reduced in patients with ALF. Time-scan experiment ( Figure 1The K-1M results showed that the loss modulus (G″), representing viscous dissipation, was significantly increased in ALF-treated livers, while the loss tangent (tanδ) was decreased, indicating that ALF shifts the viscoelastic balance of the liver towards a more elastic, solid-like state.

[0037] RNA-seq analysis results are as follows: Figure 2 As shown. Gene set enrichment analysis (GSEA, Figure 2 A) shows that mechanosensory-related pathways (such as actin cytoskeleton regulation, focal adhesion, RhoGTPase, and Hippo signaling pathways) are significantly upregulated in ALF liver. (Heatmap) Figure 2 B) showed that key mechanosensitive genes (such as Src, Palld, Rhoa, Racl, Ctgf, Cyr61) were significantly upregulated in ALF liver tissue, while metabolic function-related genes (such as A1at, Cyp1a2, Cyp3a11) were downregulated.

[0038] The above results indicate that under ALF pathological conditions, the liver transforms from a highly dissipative viscoelastic solid with rapid energy dissipation capacity into a pathological tissue with increased viscosity and sluggish mechanical response, accompanied by abnormal activation of mechanosensory pathways.

[0039] Example 2: Construction and characterization of biomimetic hydrogel microspheres with tunable viscoelasticity 1. Material preparation Alginate hydrogel precursor solution: 3% (w / v) sodium alginate (Alg) was dissolved in serum-free DMEM medium and mixed with Ca-EDTA complex solutions of different concentrations. The pH was adjusted to 7.4 with NaOH.

[0040] Decellularized matrix (dECM) pregel solution: Porcine liver tissue was subjected to three cycles of freezing at -80°C and thawing at room temperature, followed by decellularization treatment with 1% Triton X-100 / 0.1% ammonia solution for three days, washing with 1% penicillin and ultrapure water for three days, and freeze-drying to obtain dECM. A 15 mg / mL dECM solution was prepared by digestion with pepsin (dECM:pepsin = 10:1, w / w) in 0.1 M hydrochloric acid for 48 hours.

[0041] 2. Preparation of Alg-dECMIPN hydrogel microspheres Microspheres were prepared using a microfluidic device. A dECM pregel solution neutralized to pH 7.4 was mixed with an alginate precursor solution as the aqueous phase. The oil phase consisted of cyclohexane with 20% (v / v) Span 80 added. The two phases were injected at a 1:4 volumetric flow rate ratio, and the droplets were crosslinked and cured for 30 minutes at 37°C in a cyclohexane bath containing 1% (v / v) acetic acid. By adjusting the molecular weight of alginate and the calcium ion concentration, three types of microspheres with different viscoelasticities were prepared: “Fast” microspheres mimicking a healthy liver, “Slow” microspheres mimicking an ALF-prone liver, and “Elastic” microspheres with extremely weak viscoelasticity, deviating from the physiological range.

[0042] 3. Mechanical characterization and structural observation The hydrogel underwent the same compression tests and rheological analyses as in Example 1. The microstructure of the lyophilized microspheres was observed using scanning electron microscopy (SEM).

[0043] 4. Results like Figure 3 As shown, the three microspheres were designed to have comparable initial elastic modulus and storage modulus ( Figure 3 B, 3H), ensured that the biological response was primarily attributed to differences in viscoelasticity. Mechanical characterization confirmed ( Figure 3 (C-3G, 3I), "Fast" microspheres exhibited rapid stress relaxation behavior similar to that of a healthy liver; "Slow" microspheres exhibited slow stress relaxation similar to that of an ALF-affected liver; and the relaxation pattern of "Elastic" microspheres significantly exceeded the physiological range. For example... Figure 4 As shown, the prepared microspheres have a uniform particle size distribution (approximately 220 μm) and a highly interconnected porous microstructure.

[0044] Example 3: Construction of cell-hydrogel microsphere complex loaded with hADSCs and its hepatic differentiation 1. Cell load and viability assay Human adipose-derived stem cells (hADSCs) were used at a rate of 2 × 10⁻⁶. 6 Cells / mL were co-cultured for 3 days in low-adsorption 24-well plates with the three types of hydrogel microspheres prepared in Example 2. Cell viability and toxicity were assessed using a CCK-8 assay kit and a live / dead cell double staining kit.

[0045] 2. In vitro liver differentiation A standardized 23-day three-stage protocol was used to induce differentiation of hADSCs into HLCs: Pre-induction (2 days): low-glucose DMEM, supplemented with EGF (20 ng / mL) and βFGF (10 ng / mL).

[0046] Differentiation induction (7 days): low glucose DMEM, supplemented with HGF (20 ng / mL), βFGF (10 ng / mL) and NAM (0.61 mg / mL).

[0047] Maturation (14 days): Low-sugar DMEM, with added OSM (20 ng / mL), dexamethasone (1 μM) and 1×ITS supplement.

[0048] In the functional inhibition experiment, either the ROCK inhibitor Y-27632 (10 μM) or the YAP inhibitor verteporfin (0.5 μM) was added every other day throughout the differentiation cycle.

[0049] 3. Functional testing The expression of liver-specific genes (such as HNF4A, ALB, and CYP3A4) was detected by qPCR (GAPDH internal control, 2^-ΔΔCT method). HNF4α and ALB proteins were detected by immunofluorescence staining (primary antibody 48h, secondary antibody 2h, DAPI counterstaining). The activities of albumin (ALB), urea, α-1 antitrypsin (A1AT), coagulation factor VII, and CYP3A4 enzyme in the culture supernatant were detected using commercial kits.

[0050] 4. Results like Figure 5 As shown, hADSCs survived well on all three types of microspheres and were able to adhere and spread sufficiently. Figure 6-8 The results showed that HLCs differentiated in "Fast" microspheres (Fast-HLCs) had significantly higher levels of liver-specific gene and protein expression (HNF4A, ALB), metabolic function (CYP3A4 activity), and synthetic function (ALB, urea, A1AT, coagulation factor VII secretion) than HLCs in "Slow" or "Elastic" microspheres.

[0051] Example 4: Mechanism verification of the ROCK / F-actin / YAP mechanical signaling pathway 1. Cytoskeleton and YAP localization analysis Cells loaded on different microspheres were stained with F-actin (rhodamine-labeled phalloidin) and YAP immunofluorescence. The degree of F-actin polymerization, orientation, and nuclear / cytoplasmic fluorescence intensity ratio of YAP were observed and quantitatively analyzed using confocal microscopy.

[0052] 2. Results like Figure 9As shown, cells on "Fast" microspheres possessed slender, well-arranged F-actin microfilaments, with YAP primarily localized in the cytoplasm. In contrast, cells on "Slow" and "Elastic" microspheres formed coarse, disorganized stress fibers, and YAP underwent significant nuclear translocation. qPCR results showed that the expression of YAP target genes CYR61 and CTGF was significantly increased on "Slow" and "Elastic" microspheres, while their expression was significantly decreased after treatment with the ROCK inhibitor Y-27632. Figure 9 F-9G).

[0053] Figure 10 The results showed that after inhibiting ROCK or YAP using Y-27632 or verteporfen, the expression levels of liver-specific genes HNF4A and ALB, which were previously suppressed in the "Slow" and "Elastic" microspheres, were restored to the same high levels as in the "Fast" microsphere group. This indicates that the "Fast" mechanical microenvironment, which mimics the viscoelasticity of a healthy liver, promotes hepatic differentiation by inhibiting the overactivation of RhoA / ROCK signaling, thereby retaining YAP in the cytoplasm.

[0054] Example 5: Preparation and biocompatibility of JanusHA-PAMN tissue patches 1. Preparation Substrate (HA adhesive): EDC / NHS (1:1) was added to a 1% (w / v) HA solution (pH 5.0, room temperature 30 min), and dopamine hydrochloride (DA:HA = 1:1, pH 5.0, room temperature 24 h) was slowly added. The mixture was dialyzed for 72 h and then lyophilized to obtain HA-DA. A 2% (w / v) HA-DA solution was cast and dried at 60 °C to form an adhesive patch.

[0055] Top layer (PAMN non-adhesive layer): Polymerize AA (20 mmol), MA (20 mmol), NHSMA (20 mmol), and AIBN (0.104 mmol), react at 70℃ for 6 h, and purify by acetone precipitation. Mix 10 mL of LEPL (0.4 g) with 10 mL of PAMN (0.5 g) aqueous solution and dry at 60℃ to form a transparent PAMN patch.

[0056] Assembly: Cut two layers to 1cm×1cm, add 5μL of deionized water, and press at 60℃ for 5min to form.

[0057] 2. Biocompatibility assessment The patches were incubated in cell culture medium for 48 hours, and the resulting extract was used to treat hADSCs and AML-12 mouse hepatocytes. Cell viability was assessed by CCK-8 and live / dead cell staining.

[0058] 3. Results like Figure 11 As shown, HA-DA and PAMN materials were successfully synthesized. Figure 11 C). The double-layer patch can effectively adhere to the liver surface ( Figure 11 B). Biocompatibility studies confirmed that the patch was not cytotoxic to either type of cell. Figure 11 D, 11E).

[0059] Example 6: Therapeutic effect of enhanced Fast-HLCs on CCl4-induced ALF mice 1. Model Construction and Treatment A CCl4-induced ALF model was established according to the method in Example 1. 24 hours after CCl4 injection, microspheres loaded with different HLCs were adhered to the surface of mouse livers via Janus patches (n=5 / group). The sham-operated group (Sham) received CCl4 injection but did not receive treatment.

[0060] 2. Sample Collection and Analysis Twenty-four hours post-transplantation, blood and liver tissue samples were collected. Serum ALT, AST, TBiL, and ALB levels were measured. Liver tissue underwent H&E staining, TUNEL staining, and Ki67 immunofluorescence staining. The expression of liver function, antioxidant, and inflammation-related genes was detected by qPCR. Oxidative stress levels in liver tissue (MDA, O2) were assessed. - (H2O2). RNA-seq analysis was performed on some samples.

[0061] 3. Results like Figures 12-16 As shown, compared with the Sham group, serum ALT and AST levels were significantly reduced in all treatment groups, and the area of ​​liver tissue necrosis was reduced, with the Fast-HLCs group showing the most significant effect. Figure 12 B, 12C). The livers in the Fast-HLCs group had a gross appearance closer to a healthy, rosy state. Figure 12 D). TUNEL staining showed that the Fast-HLCs group had the fewest apoptotic cells, while Ki67 staining showed that it had the most proliferating cells. Figure 12 E).

[0062] qPCR results showed that Fast-HLCs treatment most effectively restored liver function-related genes (Hnf4a, Cyp3a11, Cyp1a2). Figure 13 ), and most significantly reduced oxidative stress markers ( Figure 14 ), upregulate antioxidant genes (Nqo1, Ho1, Gpx1) Figure 14 Upregulation of anti-inflammatory genes (Il10, Arg1) and downregulation of pro-inflammatory genes (Tnf, Nos2, Nlrp3) Figure 15 , Figure 16 ).

[0063] Transcriptome analysis ( Figure 17 Further evidence confirms that Fast-HLCs treatment significantly reverses aberrantly activated mechanosensory pathways in ALF (such as cell-ECM interactions and RhoGTPase). Figure 17 A) restores the macroscopic mechanical properties of the liver (stress relaxation, loss modulus, tanδ) to near-healthy levels after treatment. Figure 17 C-17E).

[0064] Example 7: Therapeutic effect of functionally enhanced Fast-HLCs on hepatectomy (PHx)-induced ALF mice 1. Model Construction and Treatment A PHx-induced ALF model was established in mice by performing approximately 70% hepatectomy. Microspheres loaded with different HLCs were adhered to the proximal mesentery of mice via Janus patches (n=5 / group). The sham-operated group (Sham) underwent only hepatectomy and did not receive cell therapy.

[0065] 2. Sample Collection and Analysis Samples were collected on postoperative days 2 and 7. Survival curves were calculated. Serum ALT, AST, total bilirubin (TBiL), and albumin (ALB) levels were measured. The liver was photographed and weighed grossly, and the liver weight / body weight ratio was calculated. H&E staining, Ki67 and CD31 immunofluorescence staining were performed. The expression of proliferation-related genes (Mki67, Pcna, Cendl), mature hepatocyte marker genes (Hnf4a, Cyp3a11, Cyp1a2, GluI), and early hepatocyte marker genes (Afp) were detected by qPCR.

[0066] 3. Results like Figures 18-23 As shown, the mice in the Fast-HLCs treatment group had the highest survival rate. Figure 18 B), serum ALT, AST, and TBiL levels were lowest, while ALB level was highest. Figure 18 C). The liver weight / body weight ratio in the Fast-HLCs group was 1.4 times that in the Sham group ( Figure 19 B), the liver is more fully formed in general ( Figure 19 A). H&E staining showed that the Fast-HLCs group had more mitotic figures and binucleated hepatocytes (A). Figure 19 C), with the highest proportion of Ki67 positive cells ( Figure 19 E, Figure 23 C).

[0067] qPCR results showed that the Fast-HLCs group contained proliferation-related genes ( Figure 19 D) and mature hepatocyte marker genes ( Figure 20 A) expression was most significantly upregulated, while the early hepatocyte marker gene Afp ( Figure 20 B) was significantly reduced, indicating that regenerated hepatocytes matured more rapidly. Simultaneously, Fast-HLCs treatment also significantly reduced oxidative stress (B). Figure 21 ) and inflammatory response ( Figure 22 ).

[0068] In summary, this invention, by revealing key changes in liver viscoelasticity during the course of ALF, proposes and validates for the first time a "mechanical preconditioning" strategy. By constructing Alg-dECMIPN hydrogel microspheres that precisely mimic the viscoelasticity of healthy livers, and using these microspheres as a mechanical preconditioning platform to induce hADSC differentiation, the resulting Fast-HLCs significantly outperformed cells prepared by traditional methods in terms of functional maturity, stability, and therapeutic efficacy. Its mechanism of action lies in providing a rapidly stress-relaxed mechanical environment, inhibiting the ROCK / YAP signaling pathway, thereby endowing cells with enhanced function and enabling them to actively correct the pathological mechanical microenvironment. This invention provides a novel, efficient, and safe technical solution for cell therapy, particularly for the treatment of severe liver diseases such as ALF.

[0069] In the description of this specification, references to terms such as "an embodiment," "example," and "specific example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0070] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for enhancing cell therapy function through mechanical pretreatment, characterized in that, The method includes: culturing and / or differentiating cells in a biomimetic mechanical microenvironment, wherein the viscoelastic properties of the biomimetic mechanical microenvironment are matched with the viscoelastic properties of a target healthy liver.

2. The method for enhancing cell therapy function through mechanical pretreatment according to claim 1, characterized in that, The target healthy tissue is the liver, and the biomimetic mechanical microenvironment is characterized by: under compression testing, its stress relaxation percentage at 60 seconds is 40% to 70%, and / or its stress relaxation half-life is 5 to 25 seconds; under shear rheological testing, its loss tangent is 0.2 to 0.

6.

3. The method for enhancing cell therapy function through mechanical pretreatment according to claim 1, characterized in that, The biomimetic mechanical microenvironment is provided by a hydrogel material with an interpenetrating polymer network structure; preferably, the hydrogel material is composed of alginate and decellularized matrix; more preferably, the hydrogel is in the form of microspheres with a diameter of 200 μm to 250 μm.

4. The method for enhancing cell therapy function through mechanical pretreatment according to claim 1, characterized in that, The cells to be treated are human adipose-derived stem cells, preferably human adipose-derived stem cells; the method further includes inducing the cells to differentiate into hepatocyte-like cells using a hepatic differentiation-inducing culture medium in the biomimetic mechanical microenvironment.

5. The method for enhancing cell therapy function through mechanical pretreatment according to claim 1, characterized in that, The method modulates the activity of the intracellular ROCK / F-actin / YAP mechanotransmission pathway by controlling the viscoelasticity of the biomimetic mechanical microenvironment; preferably, it promotes cell functional maturation by providing a rapid stress relaxation mechanical microenvironment that matches that of a healthy liver, thereby inhibiting the activation of ROCK signaling and the nuclear translocation of YAP.

6. The functionally enhanced cell-hydrogel microsphere complex prepared by the method according to any one of claims 1-5, characterized in that, The complex comprises: (a) Hydrogel microspheres with tunable viscoelasticity serving as a biomimetic mechanical microenvironment; and (b) Cells loaded on or inside the surface of the hydrogel microspheres, wherein the cells have enhanced functional maturity and stability after the mechanical pretreatment, and the cells are hepatocyte-like cells differentiated from human adipose-derived stem cells.

7. A tissue patch for cell transplantation, characterized in that, The tissue patch includes: (a) an adhesive underlayer for adhering the tissue patch to the surface of the liver; and (b) A functional layer comprising the cell-hydrogel microsphere complex of claim 6; Preferably, the adhesive underlayer is composed of dopamine-modified hyaluronic acid hydrogel, and the functional layer is a polyamino polymer-poly(acrylic acid-methacrylic acid-N-(methacryloylamino)succinimide) copolymer hydrogel patch.

8. The use of the cell-hydrogel microsphere complex of claim 6 or the tissue patch of claim 7 in the preparation of a drug for treating acute liver failure.

9. The application according to claim 8, characterized in that, The liver disease is acute liver failure or a liver disease requiring liver regeneration and functional reconstruction; the application includes: transplanting the functionally enhanced cell-hydrogel microsphere complex or the tissue patch to the site of liver injury to supplement liver function, downregulate abnormally activated YAP signaling in the host liver, reduce oxidative stress levels, inhibit inflammatory responses, and promote liver tissue regeneration.

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