CN106577637A - Preservation method of human amniotic mesenchymal stem cells - Google Patents
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- 238000004321 preservation Methods 0.000 title claims description 10
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
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Abstract
本发明涉及一种人羊膜间充质干细胞的保存方法，属于生物技术领域。人羊膜间充质干细胞保存液，由下述成分组成：枸橼酸钠(C6H5Na3O7·2H2O)12‑15g，枸橼酸(C6H8O7·H2O)3‑6g，葡萄糖(C6H12O6·H2O)12‑16g，人血浆10ml，人血白蛋白10g，水990ml。本发明的优点是：本发明提供的人羊膜间充质干细胞保存液不含动物血清，具有高安全性。实验证明，人羊膜间充质干细胞在上述保存液中在4℃保存24小时，仍表达如下三种间充质干细胞细胞膜分子：人白细胞分化抗原CD73、人白细胞分化抗原CD90和人白细胞分化抗原CD105。
The invention relates to a method for preserving human amniotic mesenchymal stem cells, belonging to the field of biotechnology. Human amniotic mesenchymal stem cell preservation solution consists of the following components: sodium citrate (C 6 H 5 Na 3 O 7 2H 2 O) 12‑15g, citric acid (C 6 H 8 O 7 H 2 O) 3-6g, glucose (C 6 H 12 O 6 ·H 2 O) 12-16g, human plasma 10ml, human albumin 10g, water 990ml. The invention has the advantages that: the human amniotic mesenchymal stem cell preservation solution provided by the invention does not contain animal serum and has high safety. Experiments have proved that human amniotic mesenchymal stem cells are stored in the above preservation solution at 4°C for 24 hours, and still express the following three mesenchymal stem cell membrane molecules: human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte differentiation antigen CD105 .
Description
技术领域technical field
本发明涉及一种人羊膜间充质干细胞的保存方法，属于生物技术领域。The invention relates to a method for preserving human amniotic mesenchymal stem cells, belonging to the field of biotechnology.
背景技术Background technique
人羊膜干细胞具有高度自我更新、增殖、可植入性和多系分化能力，异基因个体移植后无免疫排斥和致瘤性风险。而且，人羊膜干细胞来源于产妇分娩后废弃的胎盘，应用到临床不会带来医学伦理争议。羊膜干细胞具有明显的集落形成能力、增殖能力、胚胎干性、免疫调节能力及无异基因移植排斥反应与致瘤性等优良生物学特性，可用来进行诸多疾病的细胞治疗、组织器官损伤的修复与重建，是再生医学领域理想的种子细胞资源，具有广阔的临床应用前景。Human amniotic stem cells have high self-renewal, proliferation, implantability and multilineage differentiation capabilities, and there is no risk of immune rejection and tumorigenicity after transplantation in allogeneic individuals. Moreover, human amniotic stem cells are derived from the discarded placenta after childbirth, and their clinical application will not bring about medical ethics controversy. Amniotic stem cells have excellent biological characteristics such as colony formation ability, proliferation ability, embryonic stemness, immune regulation ability, and no allogeneic transplantation rejection and tumorigenicity. They can be used for cell therapy of many diseases and repair of tissue and organ damage. It is an ideal seed cell resource in the field of regenerative medicine and has broad prospects for clinical application.
近些年来，大量的研究表明，羊膜干细胞，既具有向同一胚层不同类型细胞分化的多向分化潜能，又有跨系跨胚层分化的能力，展示出良好的可塑性。如，起源中胚层的细胞既能向成骨、软骨、脂肪等中胚层细胞分化，亦可向胰腺细胞、肝细胞等内胚层细胞、神经细胞等外胚层细胞分化。In recent years, a large number of studies have shown that amniotic membrane stem cells not only have the potential to differentiate into different types of cells in the same germ layer, but also have the ability to differentiate across lineages and germ layers, showing good plasticity. For example, cells originating from the mesoderm can not only differentiate into mesoderm cells such as osteoblasts, cartilage, and fat, but also differentiate into endoderm cells such as pancreatic cells and liver cells, and ectoderm cells such as nerve cells.
人羊膜干细胞不仅具有显著的自我更新能力及跨系跨胚层多系分化潜能，且相对胚胎干细胞和其它成体干细胞，其资源丰富，易获得，无伦理争议，免疫原性低，无致瘤风险，还具分泌免疫抑制因子、神经营养因子等多种生物活性因子的强大旁分泌或自分泌功能。这些特性赋予了它在组织工程、细胞治疗、基因治疗等相关再生医学领域不可估量的临床应用价值。Human amniotic stem cells not only have significant self-renewal ability and cross-lineage, trans-germ layer and multi-lineage differentiation potential, but also have abundant resources, easy access, no ethical controversy, low immunogenicity, and no tumorigenic risk compared with embryonic stem cells and other adult stem cells. It also has a strong paracrine or autocrine function of secreting immunosuppressive factors, neurotrophic factors and other biologically active factors. These characteristics endow it with immeasurable clinical application value in tissue engineering, cell therapy, gene therapy and other related regenerative medicine fields.
发明内容Contents of the invention
本发明的第一个目的是提供人羊膜间充质干细胞保存液。The first object of the present invention is to provide human amniotic mesenchymal stem cell preservation solution.
本发明的第二个目的是提供人羊膜间充质干细胞的保存方法。The second object of the present invention is to provide a method for preserving human amniotic mesenchymal stem cells.
为实现上述目的，本发明采用以下技术方案：To achieve the above object, the present invention adopts the following technical solutions:
人羊膜间充质干细胞保存液，由下述成分组成：Human amniotic mesenchymal stem cell preservation solution consists of the following components:
枸橼酸钠(C6H5Na3O7·2H2O)12-15g，枸橼酸(C6H8O7·H2O)3-6g，葡萄糖(C6H12O6·H2O)12-16g，人血浆10ml，人血白蛋白10g，水990ml。Sodium citrate (C 6 H 5 Na 3 O 7 ·2H 2 O) 12-15g, citric acid (C 6 H 8 O 7 ·H 2 O) 3-6g, glucose (C 6 H 12 O 6 · H 2 O) 12-16g, human plasma 10ml, human serum albumin 10g, water 990ml.
所述人羊膜间充质干细胞保存液，优选由下述成分组成：The human amniotic mesenchymal stem cell preservation solution preferably consists of the following components:
枸橼酸钠(C6H5Na3O7·2H2O)13-14g，枸橼酸(C6H8O7·H2O)4-5g，葡萄糖(C6H12O6·H2O)14-15g，人血浆10ml，人血白蛋白10g，水990ml。Sodium citrate (C 6 H 5 Na 3 O 7 ·2H 2 O) 13-14g, citric acid (C 6 H 8 O 7 ·H 2 O) 4-5g, glucose (C 6 H 12 O 6 · H 2 O) 14-15g, human plasma 10ml, human serum albumin 10g, water 990ml.
所述人羊膜间充质干细胞保存液，最优选由下述成分组成：The human amniotic mesenchymal stem cell preservation solution is most preferably composed of the following components:
枸橼酸钠(C6H5Na3O7·2H2O)13.2g、枸橼酸(C6H8O7·H2O)4.8g、葡萄糖(C6H12O6·H2O)14.7g、人血浆10ml、人血白蛋白10g和水990ml。Sodium citrate (C 6 H 5 Na 3 O 7 ·2H 2 O) 13.2g, citric acid (C 6 H 8 O 7 ·H 2 O) 4.8g, glucose (C 6 H 12 O 6 ·H 2 O) 14.7g, 10ml of human plasma, 10g of human serum albumin and 990ml of water.
所述人血浆为人AB血浆。The human plasma is human AB plasma.
所述人羊膜间充质干细胞保存液的pH值为7.2-7.4。The pH value of the human amniotic mesenchymal stem cell preservation solution is 7.2-7.4.
人羊膜间充质干细胞的保存方法，包括如下步骤：将人羊膜间充质干细胞保存于上述人羊膜间充质干细胞保存液中。The method for preserving human amniotic mesenchymal stem cells comprises the following steps: preserving human amniotic mesenchymal stem cells in the above-mentioned human amniotic mesenchymal stem cell preservation solution.
所述人羊膜间充质干细胞在所述保存液中的含量是(2-3)×108个细胞/L。The content of the human amniotic mesenchymal stem cells in the preservation solution is (2-3)×10 8 cells/L.
所述保存的温度为2-8℃，最好是4℃。The storage temperature is 2-8°C, preferably 4°C.
人羊膜间充质干细胞的保存方法：将人羊膜间充质干细胞在由如下配比的物质组成的溶液中保存：枸橼酸钠(C6H5Na3O7·2H2O)13.2g、枸橼酸(C6H8O7·H2O)4.8g、葡萄糖(C6H12O6·H2O)14.7g、人AB血浆10ml、人血白蛋白10g和水990ml。Preservation method of human amniotic mesenchymal stem cells: human amniotic mesenchymal stem cells are preserved in a solution composed of the following substances: sodium citrate (C 6 H 5 Na 3 O 7 2H 2 O) 13.2g , citric acid (C 6 H 8 O 7 ·H 2 O) 4.8g, glucose (C 6 H 12 O 6 ·H 2 O) 14.7g, human AB plasma 10ml, human serum albumin 10g and water 990ml.
所述人羊膜间充质干细胞表达如下三种间充质干细胞细胞膜分子：人白细胞分化抗原CD73、人白细胞分化抗原CD90和人白细胞分化抗原CD105。不表达如下二种细胞膜分子：人白细胞分化抗原CD45和人白细胞抗原HLA-DR。The human amniotic mesenchymal stem cells express the following three mesenchymal stem cell membrane molecules: human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte differentiation antigen CD105. The following two cell membrane molecules are not expressed: human leukocyte differentiation antigen CD45 and human leukocyte antigen HLA-DR.
在上述温度和人羊膜间充质干细胞保存液中，所述保存的时间为可为12-24小时，如24小时。At the above temperature and in the human amniotic mesenchymal stem cell preservation solution, the preservation time may be 12-24 hours, such as 24 hours.
本发明的优点是：本发明提供的人羊膜间充质干细胞保存液不含动物血清，具有高安全性。实验证明，人羊膜间充质干细胞在上述保存液中在4℃保存24小时，仍表达如下三种间充质干细胞细胞膜分子：人白细胞分化抗原CD73、人白细胞分化抗原CD90和人白细胞分化抗原CD105。The invention has the advantages that: the human amniotic mesenchymal stem cell preservation solution provided by the invention does not contain animal serum and has high safety. Experiments have proved that human amniotic mesenchymal stem cells are stored in the above preservation solution at 4°C for 24 hours, and still express the following three mesenchymal stem cell membrane molecules: human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte differentiation antigen CD105 .
下面结合附图和具体实施方式对本发明做详细说明，并非对本发明的限定，凡依照本申请公开文件所进行的任何本领域的等同替换，均属于本发明的保护范围。The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but not to limit the present invention. Any equivalent replacement in the field according to the disclosure documents of this application shall fall within the scope of protection of the present invention.
附图说明Description of drawings
图1A为人羊膜间充质干细胞刚贴壁时的情况Figure 1A is the situation when human amniotic mesenchymal stem cells just adhered to the wall
图1B为人羊膜间充质干细胞开始生长时的情况Figure 1B is the situation when human amniotic mesenchymal stem cells begin to grow
图1C为人羊膜间充质干细胞增殖后的情况Figure 1C is the situation after the proliferation of human amniotic mesenchymal stem cells
图2A为人羊膜间充质干细胞通过流式细胞仪检测人白细胞分化抗原CD73的表达情况Figure 2A shows the expression of human leukocyte differentiation antigen CD73 detected by flow cytometry in human amniotic mesenchymal stem cells
图2B为人羊膜间充质干细胞通过流式细胞仪检测人白细胞分化抗原CD90的表达情况Figure 2B shows the expression of human leukocyte differentiation antigen CD90 detected by flow cytometry in human amniotic mesenchymal stem cells
图2C为人羊膜间充质干细胞通过流式细胞仪检测人白细胞分化抗原CD105的表达情况Figure 2C shows the expression of human leukocyte differentiation antigen CD105 detected by flow cytometry in human amniotic mesenchymal stem cells
图2D为人羊膜间充质干细胞通过流式细胞仪检测人白细胞分化抗原CD45的表达情况Figure 2D shows the expression of human leukocyte differentiation antigen CD45 detected by flow cytometry in human amniotic mesenchymal stem cells
图2E为人羊膜间充质干细胞通过流式细胞仪检测人白细胞抗原HLA-DR的表达情况Figure 2E shows the expression of human leukocyte antigen HLA-DR detected by flow cytometry in human amniotic mesenchymal stem cells
图3A为人羊膜间充质干细胞在人羊膜间充质干细胞保存液中4℃保存24小时后通过流式细胞仪检测人白细胞分化抗原CD73的表达情况Figure 3A shows the expression of human leukocyte differentiation antigen CD73 detected by flow cytometry after human amniotic mesenchymal stem cells were stored in human amniotic mesenchymal stem cell preservation solution at 4°C for 24 hours
图3B为人羊膜间充质干细胞在人羊膜间充质干细胞保存液中4℃保存24小时后通过流式细胞仪检测人白细胞分化抗原CD90的表达情况Figure 3B shows the expression of human leukocyte differentiation antigen CD90 detected by flow cytometry after human amniotic mesenchymal stem cells were stored in human amniotic mesenchymal stem cell preservation solution at 4°C for 24 hours
图3C为人羊膜间充质干细胞在人羊膜间充质干细胞保存液中4℃保存24小时后通过流式细胞仪检测人白细胞分化抗原CD105的表达情况Figure 3C shows the expression of human leukocyte differentiation antigen CD105 detected by flow cytometry after human amniotic mesenchymal stem cells were stored in human amniotic mesenchymal stem cell preservation solution at 4°C for 24 hours
图3D为人羊膜间充质干细胞在人羊膜间充质干细胞保存液中4℃保存24小时后通过流式细胞仪检测人白细胞分化抗原CD45的表达情况Figure 3D shows the expression of human leukocyte differentiation antigen CD45 detected by flow cytometry after human amniotic mesenchymal stem cells were stored in human amniotic mesenchymal stem cell preservation solution at 4°C for 24 hours
图3E为人羊膜间充质干细胞在人羊膜间充质干细胞保存液中4℃保存24小时后通过流式细胞仪检测人白细胞抗原HLA-DR的表达情况Figure 3E shows the expression of human leukocyte antigen HLA-DR detected by flow cytometry after human amniotic mesenchymal stem cells were stored in human amniotic mesenchymal stem cell preservation solution at 4°C for 24 hours
具体实施方式detailed description
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
α-MEM培养基为Gibco公司的产品，其产品编号为：11900024；人AB血浆购自北京红十字血液中心；注射用人血白蛋白(国药准字S20030043)。α-MEM medium is a product of Gibco Company, and its product number is: 11900024; human AB plasma was purchased from Beijing Red Cross Blood Center; human albumin for injection (Guoyao Zhunzi S20030043).
人羊膜间充质干细胞专用培养基：α-MEM(Gibco，USA)培养基中添加人AB血浆和人血白蛋白，人AB血浆在该人羊膜间充质干细胞专用培养基中的浓度是10％(体积百分浓度)，人血白蛋白在该人羊膜间充质干细胞专用培养基中的浓度是1％(体积百分浓度)，pH值为7.2-7.4。Special medium for human amniotic mesenchymal stem cells: Add human AB plasma and human serum albumin to the α-MEM (Gibco, USA) medium, and the concentration of human AB plasma in the special medium for human amniotic mesenchymal stem cells is 10 % (volume percent concentration), the concentration of human serum albumin in the special medium for human amniotic mesenchymal stem cells is 1% (volume percent concentration), and the pH value is 7.2-7.4.
实施例1、人羊膜间充质干细胞保存液Embodiment 1, human amniotic mesenchymal stem cell preservation solution
一、处方1. Prescription
二、制法Second, the method
将处方量的枸橼酸钠、枸橼酸、葡萄糖、人AB血浆、人血白蛋白依次加入水中，搅拌溶解，混匀，灭菌，即得。Add the prescribed amount of sodium citrate, citric acid, glucose, human AB plasma, and human serum albumin into water in sequence, stir to dissolve, mix well, and sterilize to obtain the product.
实施例2、人羊膜间充质干细胞的分离培养Example 2. Isolation and culture of human amniotic mesenchymal stem cells
1、细胞分离1. Cell separation
(1)羊膜取自足月剖宫产健康产妇，采集后6～12h处理。(1) Amniotic membranes were collected from healthy puerperas who delivered full-term cesarean section, and were processed 6-12 hours after collection.
(2)将直径为6cm×6cm的羊膜用0.9％氯化钠溶液清洗，剪碎至约1mm×1mm×1mm，经0.1％胶原酶和0.125％的胰酶37℃消化30-60min，用α-MEM(Gibco，USA)稀释至40ml。(2) Wash the amniotic membrane with a diameter of 6cm×6cm with 0.9% sodium chloride solution, cut it to about 1mm×1mm×1mm, digest it with 0.1% collagenase and 0.125% trypsin at 37°C for 30-60min, and use α - MEM (Gibco, USA) diluted to 40ml.
(3)将上述液体先后用100目及200目滤网过滤，去未消化的组织。(3) Filter the above liquid with 100-mesh and 200-mesh filters successively to remove undigested tissues.
(4)过滤后的含细胞的液体置于50ml离心管中，配平后放置在低温离心机内，以离心力300Xg，温度4±2℃，离心8分钟。(4) The filtered cell-containing liquid is placed in a 50ml centrifuge tube, balanced and placed in a low-temperature centrifuge, and centrifuged for 8 minutes at a centrifugal force of 300Xg and a temperature of 4±2°C.
(5)停机后轻轻取出离心管置于试管架上，离心管内容物分为二部分，上层为胶原酶、胰酶和α-MEM培养基，下层为组织碎块与细胞混合物。(5) Gently take out the centrifuge tube and place it on the test tube rack after shutdown. The content of the centrifuge tube is divided into two parts, the upper layer is collagenase, trypsin and α-MEM medium, and the lower layer is the tissue fragment and cell mixture.
(6)将上层胶原酶、胰酶和α-MEM培养基吸出。(6) Aspirate the upper collagenase, trypsin and α-MEM medium.
(7)将所分离的人羊膜单个核细胞用α-MEM培养基稀释后2000r/min离心10min洗二次。(7) Dilute the isolated human amniotic mononuclear cells with α-MEM medium, then centrifuge at 2000r/min for 10min and wash twice.
2、细胞培养2. Cell culture
将分离的人羊膜单个核细胞按1×106个细胞/cm2接种于75cm2塑料培养瓶，培养于37℃，5％CO2培养箱，培养液为人羊膜间充质干细胞专用培养基。24小时后换液，弃非贴壁细胞，以后每2～3天换液；待人羊膜间充质干细胞生长到70～80％融合，用0.25％胰酶(Sigma，USA)消化(1～2分钟)，按0.8×104～1.0×104个细胞/cm2传代。每2～3天用该人羊膜间充质干细胞专用培养基中传代一次， 使细胞浓度维持在5×105～10×105个细胞/mL。每次传代培养的条件均为37℃，5％CO2。传5代得到人羊膜间充质干细胞。The isolated human amniotic mononuclear cells were inoculated into 75cm 2 plastic culture flasks at 1×10 6 cells/cm 2 and cultured in a 37°C, 5% CO 2 incubator. The culture medium was a special medium for human amniotic mesenchymal stem cells. Change the medium after 24 hours, discard the non-adherent cells, and change the medium every 2-3 days thereafter; when the human amniotic mesenchymal stem cells grow to 70-80% confluence, digest with 0.25% trypsin (Sigma, USA) (1-2 minutes), passaging at 0.8×10 4 -1.0×10 4 cells/cm 2 . Subculture once every 2 to 3 days in the special medium for human amniotic mesenchymal stem cells, so that the cell concentration is maintained at 5×10 5 to 10×10 5 cells/mL. The conditions for each subculture were 37°C, 5% CO 2 . Human amniotic mesenchymal stem cells were obtained by passage for 5 generations.
图1A为人羊膜间充质干细胞刚贴壁时的情况，为形态呈细小的圆形。Fig. 1A is the situation of the human amniotic mesenchymal stem cells just attached to the wall, and the shape is small and round.
图1B为人羊膜间充质干细胞开始生长时的情况，48小时后，细胞开始增殖，向长梭形转变。Figure 1B shows the situation when human amniotic mesenchymal stem cells begin to grow. After 48 hours, the cells begin to proliferate and transform into long spindles.
图1C为人羊膜间充质干细胞增殖后的情况，一周后细胞开始增殖形成大小不等的细胞集落。Figure 1C is the condition of human amniotic mesenchymal stem cells after proliferation, and the cells began to proliferate to form cell colonies of different sizes after one week.
实施例3：流式细胞检测Example 3: Flow Cytometry
一、方法1. Method
实施例2得到的人羊膜间充质干细胞，用抗人CD73单克隆抗体(BioLegend，USA)通过流式细胞仪检测人白细胞分化抗原CD73的表达情况，用抗人CD90单克隆抗体(BioLegend，USA)通过流式细胞仪检测人白细胞分化抗原CD90的表达情况，用抗人CD105单克隆抗体(BioLegend，USA)通过流式细胞仪检测人白细胞分化抗原CD105的表达情况，用抗人CD45单克隆抗体(BioLegend，USA)通过流式细胞仪检测人白细胞分化抗原CD45的表达情况，用抗人白细胞抗原HLA-DR单克隆抗体(BioLegend，USA)通过流式细胞仪(型号FACSCalibur342975，BD公司)检测人白细胞抗原HLA-DR的表达情况。For the human amniotic mesenchymal stem cells obtained in Example 2, the expression of the human leukocyte differentiation antigen CD73 was detected by flow cytometry with an anti-human CD73 monoclonal antibody (BioLegend, USA), and the expression of the human leukocyte differentiation antigen CD73 was detected with an anti-human CD90 monoclonal antibody (BioLegend, USA). ) by flow cytometry to detect the expression of human leukocyte differentiation antigen CD90, use anti-human CD105 monoclonal antibody (BioLegend, USA) to detect the expression of human leukocyte differentiation antigen CD105 by flow cytometry, and use anti-human CD45 monoclonal antibody (BioLegend, USA) was used to detect the expression of human leukocyte differentiation antigen CD45 by flow cytometry, and anti-human leukocyte antigen HLA-DR monoclonal antibody (BioLegend, USA) was used to detect human The expression of leukocyte antigen HLA-DR.
二、结果2. Results
结果如图2A、图2B、图2C、图2D和图2E所示，表明该人羊膜间充质干细胞表达人白细胞分化抗原CD73、人白细胞分化抗原CD90和人白细胞分化抗原CD105。不表达人白细胞分化抗原CD45和人白细胞抗原HLA-DR。流式细胞仪检测结果表明该人羊膜间充质干细胞的纯度达到94.3-95.2％。The results are shown in Figure 2A, Figure 2B, Figure 2C, Figure 2D and Figure 2E, indicating that the human amniotic mesenchymal stem cells expressed human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte differentiation antigen CD105. Does not express human leukocyte differentiation antigen CD45 and human leukocyte antigen HLA-DR. The results of flow cytometry showed that the purity of the human amniotic mesenchymal stem cells reached 94.3-95.2%.
实施例4：人羊膜间充质干细胞的保存Example 4: Preservation of human amniotic mesenchymal stem cells
一、材料：1. Materials:
1、实施例2得到的人羊膜间充质干细胞1. Human amniotic mesenchymal stem cells obtained in Example 2
2、人羊膜间充质干细胞保存液，按实施例1制备2. Human amniotic mesenchymal stem cell preservation solution, prepared according to Example 1
二、方法：2. Method:
将实施例2得到的人羊膜间充质干细胞加入实施例1制备的人羊膜间充质干细胞保存液中，该保存液的pH值是7.2-7.4。人羊膜间充质干细胞在该人羊膜间 充质干细胞保存液的含量是(2-3)×108个细胞/L。The human amniotic mesenchymal stem cells obtained in Example 2 were added to the human amniotic mesenchymal stem cell preservation solution prepared in Example 1, and the pH value of the preservation solution was 7.2-7.4. The content of human amniotic mesenchymal stem cells in the human amniotic mesenchymal stem cell preservation solution is (2-3)×10 8 cells/L.
将接入人羊膜间充质干细胞的人羊膜间充质干细胞保存液装入血细胞保存袋中，用高频热合机切断血细胞保存袋，在4℃保存24小时。Put the human amniotic mesenchymal stem cell preservation solution inserted into the human amniotic membrane mesenchymal stem cells into the blood cell preservation bag, cut off the blood cell preservation bag with a high-frequency heat sealing machine, and preserve it at 4°C for 24 hours.
用流式细胞仪检测保存后的人羊膜间充质干细胞的如下五种人白细胞细胞膜抗原分子表达情况：人白细胞分化抗原CD73、人白细胞分化抗原CD90、人白细胞分化抗原CD105、人白细胞分化抗原CD45和人白细胞抗原HLA-DR。The expression of the following five human leukocyte membrane antigen molecules in the preserved human amniotic mesenchymal stem cells was detected by flow cytometry: human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90, human leukocyte differentiation antigen CD105, and human leukocyte differentiation antigen CD45 and human leukocyte antigen HLA-DR.
三、结果：3. Results:
结果如图3A至图3E所示，人羊膜间充质干细胞在上述人羊膜间充质干细胞保存液中在4℃保存24小时，仍表达人白细胞分化抗原CD73、人白细胞分化抗原CD90和人白细胞分化抗原CD105。不表达人白细胞分化抗原CD45和人白细胞抗原HLA-DR。The results are shown in Figure 3A to Figure 3E, human amniotic mesenchymal stem cells were preserved in the above-mentioned human amniotic mesenchymal stem cell preservation solution at 4°C for 24 hours, and still expressed human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte Differentiation antigen CD105. Does not express human leukocyte differentiation antigen CD45 and human leukocyte antigen HLA-DR.
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