WO2022042704A1 - 脱细胞基质水凝胶及其制备方法和应用 - Google Patents

脱细胞基质水凝胶及其制备方法和应用 Download PDF

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WO2022042704A1
WO2022042704A1 PCT/CN2021/115121 CN2021115121W WO2022042704A1 WO 2022042704 A1 WO2022042704 A1 WO 2022042704A1 CN 2021115121 W CN2021115121 W CN 2021115121W WO 2022042704 A1 WO2022042704 A1 WO 2022042704A1
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decellularized
preparation
solution containing
acellular matrix
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高毅
易笑
李阳
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广东乾晖生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents

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  • the present application relates to the field of biological materials, in particular to an acellular matrix hydrogel and a preparation method and application thereof.
  • ECM extracellular matrix
  • GAG glycosaminoglycan
  • ECM hydrogels include liver, small intestinal submucosa, dermis, and bladder matrix.
  • the healthy tissue derived from human ECM is very limited, which involves ethical issues.
  • immunogenicity, the synthetic hydrogel has a single composition and poor biological tissue compatibility.
  • Animal acellular ECM hydrogels are difficult to use in clinical practice due to species differences, ethics, limited sources, complex extraction process, and low efficiency.
  • the purpose of the present application is to overcome the deficiencies of the prior art and provide an acellular matrix hydrogel with high extraction efficiency and good quality, as well as a preparation method and application thereof.
  • the first is a preparation method of acellular matrix hydrogel, including the following steps:
  • tissue block was then moved into a solution containing 3% Triton X, and stirred at 250 rmp for 2 to 3 hours at a temperature of 27°C;
  • tissue block into a solution containing 4% sodium deoxycholate, and stir at a speed of 250 rmp for 2 to 3 hours under the condition of 27°C;
  • the tissue block was moved into deionized water, and soaked at 27°C for 12-18 hours to obtain an acellular scaffold
  • the decellularized scaffold was moved into 75% alcohol, soaked for 30 minutes, then moved into a solution containing 0.1% peracetic acid, and stirred at a rotational speed of 150 rpm for 2 to 3 hours;
  • the matrix powder is moved into a solution containing pepsin hydrochloride, stirred and digested for 2-3 days to obtain a pre-gel;
  • the pregel was diluted to a preset concentration at 37°C, and the acellular matrix hydrogel was obtained after standing.
  • This application preferably uses umbilical cord as a tissue material for preparing acellular matrix hydrogels.
  • Umbilical cord is a special property of human tissue medical waste, and the source is sufficient, and does not involve species and ethical issues. If the umbilical cord can be extracted from the umbilical cord
  • Decellularized ECM hydrogels are expected to open up new avenues for the treatment of mesenchymal stem cells (MSCs), significantly improve the survival efficiency of MSCs after transplantation, specifically target damaged tissues, and reduce the risk of gene mutation and tumorigenesis.
  • Engineered organs can also be created by filling them with appropriate cells, showing great promise in the fields of tissue engineering and regenerative medicine.
  • This application takes into account the clinical GMP-grade production, and the chemicals and reagents used in each step of hydrogel production can be purchased from GMP-grade commercial products, which facilitates industrial use.
  • the method of the present application and the prepared acellular matrix hydrogel can be used in 3D culture systems of various stem cells and organoids.
  • Figure 1 is a physical diagram of the intermediate product and final product obtained by the preparation method of the acellular matrix hydrogel of the present application.
  • Figure 2 is a diagram showing the staining identification of the acellular matrix hydrogel of the present application.
  • FIG. 3 is the identification diagram of immunofluorescence staining of the acellular matrix hydrogel of the present application.
  • FIG. 4 is a graph of protein mass spectrometry analysis of the acellular matrix hydrogel of the present application.
  • Figure 5 is an example diagram of the acellular matrix hydrogel of the present application combined with a variety of cells to make liver organoids for acute liver failure transplantation.
  • FIG. 6 is a diagram of an example of using the acellular matrix hydrogel of the present application for 3D culture of stem cells.
  • the embodiment of the present application takes the umbilical cord as the main processing object, and the preparation method of the present application is also applicable to tissues from other sources.
  • the preparation method of the acellular matrix hydrogel includes the following steps:
  • the umbilical cord tissue After obtaining fresh umbilical cord tissue, the umbilical cord tissue needs to be frozen at -20°C for transport to the laboratory. Specifically, liquid nitrogen can be used for rapid freezing.
  • the tissue blocks need to be tested for viruses, bacteria, and mycoplasma to ensure that the acellular matrix hydrogels produced subsequently are free from microbial contamination. After confirming that the tissue block meets the requirements, rinse the surface blood with deionized water and remove the arteries and veins of the umbilical cord and set aside.
  • the umbilical cord tissue was transferred into a solution containing 0.02% trypsin and 0.05% ethylenediaminetetraacetic acid (EDTA) and stirred for 2h at a stirring speed of 350rmp, and it was required to keep at 37°C during the experiment.
  • EDTA ethylenediaminetetraacetic acid
  • an acellular matrix scaffold was obtained from the umbilical cord tissue, which had a white, porous appearance.
  • the acellular matrix scaffold was moved into 75% alcohol and soaked for 30min.
  • acellular matrix scaffold into a clean petri dish, flatten it, pack it in a sterile bag, and freeze it in a -80°C refrigerator for 3-4 hours.
  • the pregel needs to be neutralized with 10 ⁇ PBS and NaOH before being diluted for cell culture or organoid preparation.
  • the preparation period from the decellularization step to obtaining the decellularized matrix hydrogel does not exceed one week.
  • pancreatin, Triton X, sodium deoxycholate and peroxyacetic acid are the main ones, and ribonuclease and deoxyribonuclease are not required; the surfactants used are also milder. Mechanical agitation is based on a shaker.
  • Acellular matrix hydrogels need to meet specified physicochemical parameters before they can be used clinically.
  • the standardized identification test items and results of the acellular matrix hydrogel of the present application include:
  • Acellular matrix hydrogels exhibit a randomly oriented fibrous network-like three-dimensional structure, which is mainly composed of radially arranged collagen fibers, a hierarchical pore structure (pores within pores) of additional flexible structural proteins, and fibronectin. Collagen fibers of different thicknesses are intertwined to form a three-dimensional space, forming cavities with different pore sizes and a coarse frame-like structure, which provides a structural basis for cell adhesion.
  • HE staining hematoxylin-eosin staining, referred to as HE staining, is one of the commonly used staining methods in paraffin section technology.
  • the hematoxylin staining solution is alkaline, which mainly makes the chromatin in the nucleus and the nucleic acid in the cytoplasm violet blue; eosin is an acidic dye, which mainly makes the components in the cytoplasm and the extracellular matrix red.
  • Figure 2h ⁇ Figure 2j the acellular matrix hydrogels of the present application showed the typical pink eosinophilic staining of collagen, while the typical basophilic staining of nuclear materials was not observed, indicating the acellular matrix of the present application.
  • the matrix hydrogel was free of cells and nuclei, and had organized collagen fibers, vascular pores, and fibronectin fibers in the pore walls.
  • Masson staining refers to mixing two or three anionic dyes to make collagen fibers blue and muscle fibers red. It is one of the staining methods for showing fibers in tissues. Masson's trichrome staining reconfirmed the results shown by HE staining, as shown in Fig. 2k to Fig. 2m, where the uniform blue staining indicated that the collagen was retained in the biological matrix.
  • Immunofluorescence staining was performed on the four main collagen components of the acellular matrix hydrogel and cell nuclei, wherein the cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). As shown in Figure 3, the staining results showed collagen I, IV, laminin and fibronectin staining, with little blue fluorescence of DAPI in the decellularized matrix hydrogel after decellularization.
  • the DNA content of the natural umbilical cord sample is 5160.12 ⁇ 284.27ng/mg
  • the DNA content of the decellularized sample is 19.42 ⁇ 3.20ng/mg, indicating that the nucleic acid substances in the acellular matrix hydrogel of the present application are basically removed , in line with experimental and clinical requirements.
  • H. Protein profile analysis The acellular matrix hydrogel of the present application was subjected to three biological replicate tests, and a total of 1320 proteins were identified (see Figure 4), and then according to the unique peptide analysis, the value greater than or equal to 2 was screened to obtain 883 proteins proteins, of which about 154 proteins are believed to be derived from the ECM, and 536 proteins are believed to be derived from exosomes, confirming that the acellular matrix hydrogel has a dual role as a support structure and storage of soluble signals for absorption.
  • the results of the above standardized identification and testing items show that the acellular matrix hydrogel obtained by the preparation method of the acellular matrix hydrogel of the present application relatively completely retains the pore structure of the acellular scaffold, and the decellularization is complete.
  • the matrix hydrogels are of high quality and are especially suitable for the preparation of acellular matrix hydrogels for umbilical cords.
  • the acellular matrix hydrogel of the umbilical cord as a safe GMP process-produced material, not only provides a safe and effective carrier for the clinical in vivo transplantation of MSCs, but also is expected to be used in tissue engineering, a variety of stem cells, organoids long-term culture (as shown in Figure 5).
  • acellular matrix hydrogel with umbilical cord mesenchymal stem cells (UC-MSCs), embryonic stem cells (ES) or induced pluripotent stem cells (IPS), serum-free medium is added to culture for 1-3 days.
  • U-MSCs umbilical cord mesenchymal stem cells
  • ES embryonic stem cells
  • IPS induced pluripotent stem cells
  • serum-free medium is added to culture for 1-3 days.
  • 3D cultured cell sheets (as shown in Figure 6) can be obtained, and corresponding organoids can be obtained by adding a variety of cells to co-culture.
  • acellular matrix hydrogels can also be layered, sprayed or combined with other scaffold materials as drug carriers, or repopulated with appropriate cells or applied as biological links for 3D bioprinting to create engineered organs. It is expected that various carrier materials for stem cell transplantation can also be used.
  • the present application utilizes the quadruple scheme of pancreatin, Triton X, sodium deoxycholate and peroxyacetic acid to efficiently extract acellular matrix hydrogel from umbilical cord tissue, with short preparation time and maximum retention.
  • the integrity of the internal structure of the acellular matrix provides the basis for the application of the acellular matrix hydrogel.

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Abstract

一种脱细胞基质水凝胶的制备方法，包括以下步骤：获得组织块；将所述组织块移入含0.02％的胰酶和0.05％的乙二胺四乙酸的溶液中搅拌2～3h；再将所述组织块移入含3％的Triton X的溶液中搅拌2～3h；再将所述组织块移入含4％的脱氧胆酸钠的溶液中搅拌2～3h；将所述组织块移入去离子水中浸泡12～18h，获得脱细胞支架；将所述脱细胞支架移入75％酒精中，浸泡30min，再移入含0.1％的过氧乙酸的溶液中搅拌2～3h；将所述脱细胞支架进行冻干和研磨成基质粉末之后，将所述基质粉末移入含盐酸胃蛋白酶的溶液中搅拌消化2～3d，获得预凝胶；所述预凝胶稀释成预设浓度，静置后获得脱细胞基质水凝胶。本申请从脐带组织中高效提取脱细胞基质水凝胶，且最大程度的保留了脱细胞基质内部结构的完整性。

Description

脱细胞基质水凝胶及其制备方法和应用 技术领域
本申请涉及一种生物材料领域，尤其涉及一种脱细胞基质水凝胶及其制备方法和应用。
背景技术
在生物体内，细胞的更新和分化与不断变化的环境协调一致，这种环境的特征是通过与邻近细胞的相互作用，多种因素的时空梯度变化，这在标准的二维培养条件下很难实现。开发模仿天然组织微环境的3D培养系统成为研究热点。细胞外基质(ECM)是组织和器官的细胞分泌到胞外的复合物，分布于细胞表面和细胞间隙。其成分复杂，是结构蛋白和功能蛋白的复合体，具体包含I型胶原蛋白、III型胶原蛋白、纤维连接蛋白、层粘连蛋白、糖胺聚糖(GAG)和多种细胞生长因子等。ECM经胰蛋白酶消化后可获得ECM水凝胶，在组织工程领域所有的生物材料中，它被认为是一种理想的工具。
现有的ECM水凝胶包括：肝脏、小肠粘膜下层、真皮、膀胱基质，但是人体ECM来源的健康组织非常有限、涉及伦理问题，动物ECM来源的存在种属差异性，容易造成物种间病毒传染及免疫原性，人工合成的水凝胶成分单一，生物组织相容性差。动物脱细胞ECM水凝胶因存在物种差异，涉及伦理、来源有限，提取过程复杂、效率低等问题，难以用于临床。
发明内容
本申请的目的是，克服现有技术的不足，提供一种提取效率高、质量好的脱细胞基质水凝胶，及其制备方法和应用。
为达到以上技术目的，本申请采用的技术方案如下：
首先是一种脱细胞基质水凝胶的制备方法，包括以下步骤：
获得组织块；
将所述组织块移入含0.02％的胰酶和0.05％的乙二胺四乙酸的溶液中，在37℃的条件下，以350rmp的转速搅拌2～3h；
再将所述组织块移入含3％的Triton X的溶液中，在27℃的条件下，以250rmp的转速搅拌2～3h；
再将所述组织块移入含4％的脱氧胆酸钠的溶液中，在27℃的条件下，以250rmp的转速搅拌2～3h；
将所述组织块移入去离子水中，在27℃的条件下浸泡12～18h，获得脱细胞支架；
将所述脱细胞支架移入75％酒精中，浸泡30min，再移入含0.1％的过氧乙酸的溶液中，以150rmp的转速搅拌2～3h；
将所述脱细胞支架进行冻干和研磨成基质粉末之后，将所述基质粉末移入含盐酸胃蛋白酶的溶液中搅拌消化2～3d，获得预凝胶；
所述预凝胶在37℃的条件下稀释成预设浓度，静置后获得脱细胞基质水凝胶。
与现有技术相比较，本申请具有如下优势：
(1)本申请优选使用脐带作为制备脱细胞基质水凝胶的组织材料，脐带作为人体组织医疗废弃物的特殊属性，来源充分，不涉及种属及伦理道德问题，若能从脐带中提取出脱细胞ECM水凝胶，有望为间充质干细胞(MSCs)治疗开辟新的途径，显著提高MSCs移植后的存活效率，特异性的定向于受损组织，降低基因突变、致瘤的风险。还可以用适当的细胞填充创建工程化的器官，在组织工程学和再生医学领域，显示出巨大的应用前景。
(2)本申请确立了胰酶、Triton X、脱氧胆酸钠和过氧乙酸四联方案，从脐带组织中高效提取脱细胞ECM水凝胶，仅需1-2d时间可获得高质量的脱细胞支架，6-7d时间即可获得水凝胶，且最大程度的保留了ECM内部结构的完整性。
(3)本申请考虑到临床的GMP级生产，在水凝胶生产的每个步骤中使用的化学药品和试剂都可以在GMP级商品处购得，为产业化使用提供便利。
(4)本申请的方法及所制备的脱细胞基质水凝胶可以用于多种干细胞以及类器官的3D培养系统。
附图说明
图1为采用本申请的脱细胞基质水凝胶制备方法所获得的中间产物和最 终产物的实物图。
图2为本申请的脱细胞基质水凝胶的染色鉴定图。
图3为本申请的脱细胞基质水凝胶的免疫荧光染色鉴定图。
图4为本申请的脱细胞基质水凝胶的蛋白质质谱分析图。
图5为本申请的脱细胞基质水凝胶结合多种细胞做成肝脏类器官，用于急性肝衰竭移植中的实例图。
图6为采用本申请的脱细胞基质水凝胶用于干细胞3D培养的实例图。
具体实施方式
以下结合附图和具体实施方式对本申请作进一步详细描述。
实施例一
脱细胞基质水凝胶的制备方法
本申请的实施例以脐带为主要处理对象，其他来源的组织也同样适用本申请的制备方法。脱细胞基质水凝胶的制备方法包括以下步骤：
(1)获得组织块；
获得新鲜的脐带组织之后，需要将脐带组织在-20℃的条件下冻存以便于运送至实验室，具体可以使用液氮进行快速冻存。下一步，需要对组织块进行病毒、细菌、支原体检测，以保证后续制成的脱细胞基质水凝胶无微生物污染。确认组织块符合要求之后，用去离子水冲洗表面血液并去除脐带的动脉和静脉，备用。
(2)组织块脱细胞：
a)将脐带剪成3-4cm的小段(见图1a)常温下在放入去离子水中搅拌3次，每次1h，300rmp。
b)脐带组织移入含0.02﹪的胰酶和0.05﹪的乙二胺四乙酸(EDTA)溶液中搅拌2h搅拌速度350rmp，实验中需要一直保持37℃。
c)去离子水清洗脐带3次，每次10min。
d)把脐带放在含3﹪的Triton X溶液中在摇床上搅拌2h，速度250rmp，温度为27.0℃。
e)去离子水清洗脐带3次，每次10min。
f)把脐带放在含4﹪的脱氧胆酸钠溶液中，在摇床上搅拌2.5h，转速250rmp，温度为27.0℃。
g)过量的去离子水冲洗脐带15min，再用去离子水浸泡12h，温度为27.0℃。
如图1b所示，从脐带组织获得脱细胞基质支架，该脱细胞基质支架呈洁白的，多孔的外观。
(3)脱细胞基质支架消毒：
此后的操作均需在超净台内进行。
h)把脱细胞基质支架移入75％酒精浸泡30min。
i)去离子水清洗脐带3次，每次10min。
j)移入含0.1﹪的过氧乙酸溶液中搅拌2-3h，速度为150rpm。
k)然后转移至1×PBS溶液(磷酸缓冲液)中搅拌，浸泡15min，更换去离子水，重复3-5次。
(4)脱细胞基质支架冷冻、干燥和研磨：
a)将脱细胞基质支架放入干净培养皿中，铺平，无菌袋装好，放入-80℃冰箱冷冻3-4h。
b)待其完全冻结后将脱细胞基质支架放入冻干机里冻干。取出冻干后的脱细胞基质支架剪碎，放入高压灭菌后的研磨钢罐中，液氮环境下预冷15min左右，在研磨器中研磨(60Hz、120s)。从研磨钢罐中取出的基质粉末(见图1c和图1d)。
(5)脱细胞基质支架消化：
在100ml盐酸胃蛋白酶溶液中消化2-3d以后，即配制成10mg/ml预凝胶(见图1e)，可以置于4℃进行无菌保存，后续使用时，稀释成预设的浓度，即可获得脱细胞基质水凝胶。
进一步地，所述预凝胶稀释以准备用于进行细胞培养或者制备类器官之前，需要使用10×PBS和NaOH进行中和。
综上，从脱细胞步骤到获得脱细胞基质水凝胶，制备周期不超过一周。从所使用的试剂来看，以胰酶、Triton X、脱氧胆酸钠和过氧乙酸为主，不需要使用核糖核酸酶和脱氧核糖核酸酶；所使用的表面活性剂也较温和。机械搅拌以摇床为主。
实施例二
脱细胞基质水凝胶的标准化鉴定
脱细胞基质水凝胶需要符合指定的理化指标，才能应用于临床。具体地，对本申请的脱细胞基质水凝胶的标准化鉴定检测项目及其结果包括：
A.成胶实验：在37℃的条件下，将所述预凝胶配平后室温静置10min即成水凝胶(见图1f)，水凝胶呈白色半透明的三维立体凝状物，可塑性极强。
B.产量：3个批次10根脐带脱细胞支架冻干后总重量为16.6g，研磨后平均每根脐带可以获得冻干粉末1.66g，可做成160ml预凝胶。
C.扫描电镜检查：样本为团块状，扫描电镜下观察可见超微结构在加工后仍保存良好，即使在高放大倍率下，纳米纤维胶原蛋白结构也完好无损(见图1g)，证实了该方案提取脐带脱细胞基质水凝胶的有效性，说明本申请所提供的制备方法对脱细胞基质支架造成的损伤很小。脱细胞基质水凝胶呈随机取向的纤维网状立体结构，主要由径向排列的胶原纤维、附加的柔性结构蛋白的分层孔结构(孔中的孔)、纤连蛋白组成。粗细不等的胶原纤维相互交织构成立体空间，形成孔径不同的空洞，粗大的框架式结构，为细胞的粘附提供了结构基础。
D.HE染色：苏木精-伊红染色法(hematoxylin-eosin staining)，简称HE染色法，石蜡切片技术里常用的染色法之一。苏木精染液为碱性，主要使细胞核内的染色质与胞质内的核酸着紫蓝色；伊红为酸性染料，主要使细胞质和细胞外基质中的成分着红色。如图2h～图2j所示，本申请的脱细胞基质水凝胶显示了胶原蛋白典型的粉红色嗜酸性染色，而未观察到细胞核材料典型的嗜碱性染色，说明了本申请的脱细胞基质水凝胶无细胞及细胞核残留，并且可见有组织的胶原纤维、血管孔、在孔壁内还包含纤连蛋白纤维。
E.Masson染色：Masson染色是指用两种或三种阴离子染料混合，使得胶原纤维呈蓝色，肌纤维呈红色，是显示组织中纤维的染色方法之一。Masson的三色染色再次证实了如HE染色所示的结果，如图2k～图2m所示，均匀的蓝色染色表明胶原蛋白保留在生物基质中。
F.免疫荧光染色：对脱细胞基质水凝胶的四种主要胶原成分及细胞核进行免疫荧光颜色，其中细胞核采用DAPI(4',6-二脒基-2-苯基吲哚)染色。如图3所示，染色结果显示出了胶原I、IV，层粘连蛋白和纤连蛋白染色，脱细胞之后脱细胞基质水凝胶中几乎不存在DAPI的蓝色荧光。
G.dsDNA含量测定；天然脐带样品DNA含量为5160.12±284.27ng/mg，脱细胞样品中DNA含量为19.42±3.20ng/mg，表明本申请的脱细胞基 质水凝胶中的核酸物质基本被去除，符合实验和临床要求。
H.蛋白质谱分析：对本申请的脱细胞基质水凝胶进行三次生物学重复试验，总共鉴定到的1320个蛋白(见图4)，然后根据unique peptide分析，取值大于等于2筛选得到883个蛋白质，其中约154种蛋白质被认为是源自ECM，而536种蛋白质则被认为来自外泌体，证实了该脱细胞基质水凝胶具有作为支持结构和吸收的可溶性信号的储存的双重作用。
上述标准化鉴定检测项目的结果表明，本申请的脱细胞基质水凝胶的制备方法所获得的脱细胞基质水凝胶，较完整地保留了脱细胞支架的孔隙结构，脱细胞彻底，该脱细胞基质水凝胶的质量高，尤其适用于制备脐带的脱细胞基质水凝胶。脐带的脱细胞基质水凝胶作为一种安全的GMP工艺化生产的材料，不仅为临床上MSCs的体内移植提供了一种安全有效的载体，还有望用于组织工程，多种干细胞、类器官的长期培养(如图5所示)。具体地，将所述脱细胞基质水凝胶与脐带间充质干细胞(UC-MSCs)、胚胎干细胞(ES)或诱导多功能干细胞(IPS)混合后，加入无血清培养基培养1-3d即可获得3D培养的细胞片(如图6所示)，加入多种细胞共同培养可获得相应的类器官。
进一步地，该脱细胞基质水凝胶还可以进行铺层、喷涂或与其他支架材料作为药物载体，或者用适当的细胞重新填充或作为生物链接应用于三维生物打印以创建工程化的器官。展望也可用于各种干细胞移植的载体材料。
综上所述，本申请利用了胰酶、Triton X、脱氧胆酸钠和过氧乙酸四联方案，从脐带组织中高效提取脱细胞基质水凝胶，制备时间短，且最大程度的保留了脱细胞基质内部结构的完整性，为脱细胞基质水凝胶的应用提供了基础。
上述实施例为本申请较佳的实施方式，但并不仅仅受上述实施例的限制，其他的任何未背离本申请的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，均包含在本申请的保护范围之内。

Claims (11)

  1. 一种脱细胞基质水凝胶的制备方法，其特征在于，包括以下步骤：
    获得组织块；
    将所述组织块移入含0.02％的胰酶和0.05％的乙二胺四乙酸的溶液中，在37℃的条件下，以350rmp的转速搅拌2～3h；
    再将所述组织块移入含3％的Triton X的溶液中，在27℃的条件下，以250rmp的转速搅拌2～3h；
    再将所述组织块移入含4％的脱氧胆酸钠的溶液中，在27℃的条件下，以250rmp的转速搅拌2～3h；
    将所述组织块移入去离子水中，在27℃的条件下浸泡12～18h，获得脱细胞支架；
    将所述脱细胞支架移入75％酒精中，浸泡30min，再移入含0.1％的过氧乙酸的溶液中，以150rmp的转速搅拌2～3h；
    将所述脱细胞支架进行冻干和研磨成基质粉末之后，将所述基质粉末移入含盐酸胃蛋白酶的溶液中搅拌消化2～3d，获得预凝胶；
    所述预凝胶在37℃的条件下稀释成预设浓度，静置后获得脱细胞基质水凝胶。
  2. 如权利要求1所述的方法，其特征在于，所述组织块来源于脐带。
  3. 如权利要求1所述的方法，其特征在于，所述组织块裁切成3～4cm宽的块段。
  4. 如权利要求1所述的方法，其特征在于，所述组织块和脱细胞支架移入不同的处理溶液之前，还包括用去离子水清洗的步骤。
  5. 如权利要求1所述的方法，其特征在于，所述将所述脱细胞支架冻干和研磨成基质粉末，包括：
    将所述脱细胞支架置入-80℃的环境3～4h；
    转移到冻干机进行冻干处理；
    转移到液氮预冷的研磨器内研磨。
  6. 如权利要求5所述的方法，其特征在于，所述研磨器的研磨频率为60Hz，研磨时间为120s。
  7. 如权利要求1所述的方法，其特征在于，所述预凝胶的浓度为10mg/ml。
  8. 如权利要求1所述的方法，其特征在于，所述预凝胶用于培养细胞和制备类器官前需利用磷酸缓冲液和氢氧化钠进行中和。
  9. 一种脱细胞基质水凝胶，其特征在于，其采用如权利要求1～8所述的脱细胞基质水凝胶的制备方法制得。
  10. 如权利要求9所述的脱细胞基质水凝胶在制备药物载体中的应用，以及/或者在制备干细胞移植载体中的应用，以及/或者在构建人工器官中的应用。
  11. 如权利要求1～8所述的脱细胞基质水凝胶的制备方法在制备药物载体中的应用，以及/或者在制备干细胞移植载体中的应用，以及/或者在构建人工器官中的应用。
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