CN-121975619-A - Kidney tubule and peritubular microenvironment simulation system based on vein-imitating network chip, construction method and application thereof

CN121975619ACN 121975619 ACN121975619 ACN 121975619ACN-121975619-A

Abstract

A system for simulating the microenvironment of kidney tubule and perivascular based on the leaf vein simulating network chip is composed of the bionic leaf vein network micro-fluidic chip, the immortalized human near-end kidney tubule epithelial cells, human umbilical vein endothelial cells and cell culture medium, and features that the cell suspension of immortalized human near-end kidney tubule epithelial cells, immortalized human near-end kidney tubule epithelial cells and human umbilical vein endothelial cells is poured in the chip, and after the cells are adhered to wall, the cell culture medium is continuously injected to simulate the liquid flowing microenvironment in human body to obtain a corresponding simulating system.

Inventors

Dou meng
Nishigaya Kaoru
TIAN PUXUN
HUANG XIAOYAN
Zheng Bingxuan
WEI TIAN

Assignees

西安交通大学医学院第一附属医院

Dates

Publication Date: 20260505
Application Date: 20260205

Claims (9)

1. A kidney tubule and perivascular microenvironment simulation system based on a simulated vein network chip is characterized by comprising a simulated vein network microfluidic chip, immortalized human proximal kidney tubule epithelial cells, human umbilical vein endothelial cells and a cell culture medium, wherein a microfluidic channel network of the simulated vein network microfluidic chip is of a kidney tubule structure, a cell layer is attached to the inner wall of a tube cavity of the microfluidic channel network, when the cell layer is the immortalized human proximal kidney tubule epithelial cells, a kidney tubule simulation system is formed, when the cell layer is a cell suspension of the immortalized human proximal kidney tubule epithelial cells and human umbilical vein endothelial cells, a kidney tubule perivascular microenvironment simulation system is formed, and then the cell activity in the kidney tubule simulation system and the kidney tubule perivascular microenvironment simulation system is maintained by continuously pouring fresh cell culture medium into the simulated vein network microfluidic chip and discharging the metabolized old cell culture medium, wherein the quantity ratio of the immortalized human proximal kidney tubule epithelial cells and the human umbilical vein endothelial cells in the kidney tubule perivascular microenvironment simulation system is 1:2.
2. A construction method of a tubular and peritubular microenvironment simulation system based on a vein-simulated network chip is characterized by comprising the following steps: step S1, preprocessing a bionic vein network micro-fluidic chip; S2, assembling the preprocessed bionic vein network micro-fluidic chip on a fixing device, and connecting one end of a liquid inlet pipe and one end of a liquid outlet pipe to a liquid inlet and a liquid outlet in a micro-fluidic channel network of the bionic vein network micro-fluidic chip respectively, wherein the other end of the liquid inlet pipe is connected with an injector which is detachably clamped on a microinjection pump, and the other end of the liquid outlet pipe is connected to a recovery cup so as to complete the assembly of the whole biological reaction device; S3, placing the assembled biological reaction device under an ultraviolet lamp for irradiation, and flushing a bionic vein network micro-fluidic chip by using sterile PBS (phosphate buffer solution) for sterilization treatment; Step S4, after the biological reaction device is sterilized, target cells are injected into a microfluidic channel network of a bionic vein network microfluidic chip through a microinjection pump, the target cells flow out from a cup to be recovered, the target cells are completely poured, the biological reaction device after the target cells are completely poured is transferred to a cell culture box and kept stand to enable the target cells to be attached, after the target cells are attached, fresh cell culture media are continuously injected into the microfluidic channel network of the bionic vein network microfluidic chip at a preset flow rate through the microinjection pump, and the metabolized old cell culture media are discharged through a liquid outlet of the microfluidic channel network to realize dynamic replacement of the cell culture media, simulate in-vivo liquid flowing microenvironment and maintain in-vitro activity of the target cells, when the target cells are only immortalized human proximal renal tubular epithelial cells, the simulation system is a renal tubular simulation system, and when the target cells are cell suspension of the immortalized human proximal renal tubular epithelial cells and human umbilical vein endothelial cells with the quantity ratio of 1:2.
3. The method for constructing a tubular and peritubular microenvironment simulation system based on a simulated vein network chip as claimed in claim 2, wherein the simulated vein network micro-fluidic chip comprises a PDMS vein network lower chip and a PDMS upper cover plate laminated and bonded with the PDMS vein network lower chip.
4. The method for constructing a system for simulating the microenvironment of the renal tubules and the periphery of the tubular ducts based on the simulated vein network chip as set forth in claim 3, wherein the method for preparing the lower chip of the PDMS vein network specifically comprises the following steps: Washing fresh osmanthus fragrans leaves with deionized water, soaking the washed fresh osmanthus fragrans leaves in a NaOH solution with the mass fraction of 3.5-5wt%, heating to a boiling state, maintaining for 30-40 min, taking out, and removing residual mesophyll tissues to obtain a leaf vein network skeleton; The method comprises the steps of photographing a vein network skeleton specimen to obtain a digital high-definition picture of the vein network skeleton, importing the digital high-definition picture of the vein network skeleton into LEAF GUI software, processing the vein network skeleton into black by using an image binarization algorithm in the LEAF GUI software, processing non-runner areas outside the vein network skeleton into white to obtain a binarized image of the vein network skeleton, observing whether the skeleton of the vein network skeleton specimen is complete or not, sputtering a 400-500 nm chromium coating on the vein network skeleton specimen to obtain chromed vein if the skeleton structure is complete, and preparing the vein network skeleton specimen by re-selecting fresh osmanthus fragrans leaves if the skeleton structure is incomplete; spin-coating hexamethyldisilane on a silicon wafer, curing to form a hexamethyldisilane coating with the thickness of 3-4 mu m, spin-coating a photosensitive polymer on the surface of the hexamethyldisilane coating, curing to form a photosensitive polymer coating with the thickness of 3-4 mu m, placing the silicon wafer spin-coated with the hexamethyldisilane coating and the photosensitive polymer coating in an exposure station, taking chromed veins as a photomask and aligning with the surface of the silicon wafer, irradiating the silicon wafer by ultraviolet rays, and then placing the silicon wafer after the ultraviolet rays are irradiated into a NaOH solution with the mass fraction of 0.5-1wt%, and decomposing the photosensitive polymer in the NaOH solution to obtain the silicon wafer with vein network negative polymer patterns; Uniformly sputtering a chromium layer with the thickness of 150-200 nm on a silicon wafer with a vein network negative polymer pattern, immersing the silicon wafer into an acetone solution to dissolve the vein network negative polymer pattern after the chromium layer is deposited, and obtaining a chromed silicon wafer; And (3) carrying out first vacuum degassing treatment on the mixed solution of the polydimethylsiloxane prepolymer and the curing agent according to the mass ratio of (9-10), carrying out alkylation treatment on the silicon wafer vein mould with the vein network negative polymer pattern, casting the mixed solution of the polydimethylsiloxane prepolymer and the curing agent after the first vacuum degassing treatment on the silicon wafer vein mould after the alkylation treatment, carrying out second vacuum degassing treatment, drying and curing the mixed solution of the polydimethylsiloxane prepolymer and the curing agent in the silicon wafer vein mould after the second vacuum degassing treatment, and stripping the silicon wafer vein mould with the vein network negative polymer pattern after the mixed solution of the polydimethylsiloxane prepolymer and the curing agent is cured and molded to obtain the PDMS vein network lower chip with the vein network negative polymer pattern.
5. The method for constructing a system for simulating the microenvironment of a renal tubule and a peritubular vein based on a vein-like network chip as set forth in claim 3, wherein the method for preparing the PDMS upper cover sheet comprises the following steps: And (3) carrying out first vacuum degassing treatment on the mixed solution of the polydimethylsiloxane prepolymer and the curing agent according to the mass ratio of (9-10), casting the mixed solution of the polydimethylsiloxane prepolymer and the curing agent after the first vacuum degassing treatment in a cell culture dish, carrying out second vacuum degassing treatment, drying and curing the mixed solution of the polydimethylsiloxane prepolymer and the curing agent in the cell culture dish after the second vacuum degassing treatment, and stripping the cell culture dish after the mixed solution of the polydimethylsiloxane prepolymer and the curing agent is cured and molded to obtain the PDMS upper cover plate.
6. The method for constructing a system for simulating the microenvironment of the renal tubules and the periphery of the renal tubules based on the vein-like network chip as set forth in claim 2, wherein the method for preparing the immortalized human proximal renal tubular epithelial cells specifically comprises the following steps: a) Resuscitating primary human kidney epithelial cells, re-suspending the primary human kidney epithelial cells by using a DMEM/F12 complete medium which is preheated to 37 ℃, inoculating the primary human kidney epithelial cells into a cell culture dish according to a cell density of 2-4 x 10-6, and culturing the primary human kidney epithelial cells in a cell culture box with 37 ℃ and 5% CO 2 , wherein the DMEM/F12 complete medium comprises 90% DMEM/F12 basal medium, 10% fetal bovine serum and 0.5-1% penicillin-streptomycin diab; b) When primary human kidney epithelial cells grow to 70-80% confluence, discarding an old DMEM/F12 complete medium, replacing the old DMEM/F12 complete medium with a DMEM/F12 basic medium, and infecting the primary human kidney epithelial cells for 12-24 hours under a cell culture box with 37 ℃ and 5% CO 2 by using lentivirus SV40T and lentivirus hTERT in a combined mode, wherein the MOI value of the lentivirus SV40T is 150, the MOI value of the lentivirus hTERT is 150, and the molar ratio of the lentivirus SV40T to the lentivirus hTERT is 1:1; c) After infection, discarding the old DMEM/F12 basal medium, and screening and culturing for 48-96 h in a cell culture box containing 1ug/mL Puromycin DMEM/F12 complete medium at 37 ℃ and 5% CO 2 ; d) After screening culture, discarding the old DMEM/F12 complete medium containing Puromycin, and carrying out conventional subculture by using the new DMEM/F12 complete medium, wherein the subculture is carried out once every 48-96 hours to obtain semi-immortalized human kidney epithelial cells; e) After transferring to 2-3 generations, when the semi-immortalized human kidney epithelial cells grow to 80-90% confluence, identifying the semi-immortalized human kidney epithelial cells after passage through immunofluorescence staining, obtaining immortalized human proximal renal tubule epithelial cells if the identified semi-immortalized human kidney epithelial cells successfully express rabbit polyclonal CK-18, and re-operating the steps a-e until the identified semi-immortalized human kidney epithelial cells successfully express rabbit polyclonal CK-18.
7. The method for constructing a tubular and peritubular microenvironment simulation system based on a simulated vein network chip as set forth in claim 2, wherein the step S1 specifically comprises sequentially performing plasma hydrophilization treatment, sterilization treatment and gelatin treatment on the simulated vein network microfluidic chip.
8. Use of the vein-like network chip-based tubular and peritubular microenvironment simulation system according to claim 1 or the vein-like network chip-based tubular and peritubular microenvironment simulation system constructed according to any one of claims 2-7 in vitro to simulate tubular and peritubular capillaries.
9. Use of the simulated vein network chip-based tubular and peritubular microenvironment simulation system according to claim 1 or the simulated vein network chip-based tubular and peritubular microenvironment simulation system constructed by any one of claims 2-7 for evaluating renal clearance characteristics and renal toxicity risks of a drug, wherein the drug comprises, but is not limited to, an anti-tumor drug, an antibiotic, and an immunosuppressant.

Description

Kidney tubule and peritubular microenvironment simulation system based on vein-imitating network chip, construction method and application thereof Technical Field The invention relates to the technical field of biomedical engineering and organ chips, in particular to a renal tubule and peritubular microenvironment simulation system based on a vein-imitating network chip, and a construction method and application thereof. Background Kidney disease is a significant health challenge worldwide. According to the international kidney disease society of 2023, the global chronic kidney disease median prevalence is as high as 9.5% and the related mortality is 2.4%. Currently, the progression of kidney disease is considered irreversible, end stage renal patients must rely on kidney replacement therapy, and kidney transplantation is the only means by which healing can be achieved. However, the current treatment mode faces serious dilemma in that dialysis treatment is expensive and accompanied by various complications, seriously affecting the quality of life of the patient, while kidney transplantation, which is the best solution, is limited by a serious shortage of donor organs for a long period of time. Thus, global kidney disease is heavily burdened, and there is an urgent need to develop more effective treatments or innovative kidney alternatives by delving into the mechanism of kidney injury. With the rapid development of biological tissue engineering and microfluidic technology, organ chips become an important platform for simulating the complex structure and physiological microenvironment of human organs in vitro, and are expected to innovate drug development and life science research. The technology simulates key physiological conditions in a human body by efficiently conveying nutrient substances in a micro-channel system, applying fluid shear force stimulation and constructing a three-dimensional culture environment. In the kidney field, the filtering function of glomerulus can be better simulated by various biological materials, but the simulation of complex functions such as renal tubule reabsorption still lacks an effective scheme. Despite significant advances in organ-chip technology, most of the current kidney-cell chip models suffer from too simple structure and inadequate biomimetic. In particular, challenges are faced in accurately controlling the peritubular microenvironment (e.g., higher order biochemical signaling and gradient distribution), inducing physiological levels of flow shear stress, increasing model complexity (e.g., integrating vasculature, stromal cells, or immune cells), modeling multi-organ interactions, and reducing variability of the model itself (e.g., inter-batch differences in tubular size, structure, and function). Therefore, the development of a novel bionic chip capable of highly simulating the physiological functions of kidneys, particularly the complex microenvironment of the tubules, is very important for promoting the research of the kidneys and realizing the substitution of in vitro functions. Construction of functional kidney tubule chips is highly dependent on the appropriate cell source. Currently, the cells most commonly used in such models include a variety of immortalized tubular epithelial cell lines, such as canine kidney-derived MDCK cells, porcine kidney-derived LLC-PK1 cells, and human HK-2 cells. However, none of these cell lines completely reproduces the phenotype of the primary cells and often shows insufficient functional differentiation in culture, limiting the physiological relevance of the model. Human primary proximal tubular epithelial cells are generally considered to be "gold standard" for in vitro modeling, better preserving in vivo function, but their use faces significant bottlenecks in that cell sources have significant donor individual variability, limited self-renewal and expansion capacity, resulting in low experimental reproducibility and difficulty in obtaining sufficient cells for high throughput studies. Although the number of passages can be prolonged by special treatments (e.g., using antisense nucleotides or RNA interference), the problems of donor variation and standardization are not fundamentally solved. In summary, although the microfluidic technology and organ chip concepts provide powerful tools for kidney research, and studies have been attempted to improve chip performance using biomimetic designs, such as Gershlak（Gershlak JR, Hernandez S, Fontana G, et al. Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds. Biomaterials. 2017. 125: 13-22.） et al, which originally used decellularized plant veins as a perfusable tissue engineering scaffold, demonstrating the potential of natural vein networks for delivering fluids and supporting cell growth, and domestic health-teaching teams, which successfully constructed highly simulated "tubular-interstitial" chips using microfluidic chip tissue enginee