WO2022099472A1 - 一种含有芦荟多糖包被层的羊水间充质干细胞冻存管 - Google Patents

一种含有芦荟多糖包被层的羊水间充质干细胞冻存管 Download PDF

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WO2022099472A1
WO2022099472A1 PCT/CN2020/127852 CN2020127852W WO2022099472A1 WO 2022099472 A1 WO2022099472 A1 WO 2022099472A1 CN 2020127852 W CN2020127852 W CN 2020127852W WO 2022099472 A1 WO2022099472 A1 WO 2022099472A1
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cryopreservation
mesenchymal stem
cryopreservation tube
polysaccharide
stem cells
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PCT/CN2020/127852
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English (en)
French (fr)
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孟永辉
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南通市巨久新材料科技有限公司
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Priority to PCT/CN2020/127852 priority Critical patent/WO2022099472A1/zh
Priority to DE212020000278.9U priority patent/DE212020000278U1/de
Publication of WO2022099472A1 publication Critical patent/WO2022099472A1/zh

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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
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
    • A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen

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  • the utility model belongs to the field of stem cells and relates to a stem cell cryopreservation tube, in particular to an amniotic fluid mesenchymal stem cell cryopreservation tube containing an aloe polysaccharide (Pullulan) coating layer.
  • a stem cell cryopreservation tube in particular to an amniotic fluid mesenchymal stem cell cryopreservation tube containing an aloe polysaccharide (Pullulan) coating layer.
  • amniotic fluid mesenchymal stem cell research is how to improve its activity after cryopreservation and resuscitation.
  • the purpose of the present utility model is to provide an amniotic fluid mesenchymal stem cell cryopreservation tube containing an aloe polysaccharide coating layer, so as to improve the activity of the amniotic fluid mesenchymal stem cells after cryopreservation and recovery.
  • An amniotic fluid mesenchymal stem cell cryopreservation tube comprises a cryopreservation tube body, the inner wall of the cryopreservation tube body is provided with a coating layer, and the coating layer is made of aloe vera polysaccharide material.
  • the thickness of the aloe polysaccharide coating layer is 0.4-0.5mm.
  • the inner wall of the cryopreservation tube provided by the utility model contains an aloe polysaccharide coating layer.
  • Figure 1 is a schematic structural diagram of a cryopreservation tube of the present invention, and Figure 1 is a coating layer of aloe polysaccharide.
  • step S1 aloe polysaccharide (sterilized by ⁇ -ray cobalt 60 irradiation, purchased from Shanghai Yuanye Bio-Technology Co., Ltd, article number: B24395) is dissolved in sterile water to prepare a 3 mg/mL aloe polysaccharide solution, spare;
  • Step S2 add 2 mL of the above solution into a 2 mL Corning cryopreservation tube, and let stand overnight at room temperature;
  • Step S3 after slowly pouring the solution in the freezing tube, and drying at 50° C. for 1 hour, a uniform aloe polysaccharide coating layer can be formed on the inner wall of the freezing tube, and the coating layer thickness is 0.4-0.5 mm.
  • Human amniotic fluid mesenchymal stem cells were cultured in DMEM/F12 containing 10% fetal bovine serum and 10 ng/mL b-FGF under the conditions of 37°C, 5% CO 2 , and saturated humidity.
  • RPMI 1640 medium is a cryoprotectant.
  • the experiment was divided into a coated cryopreservation group and a conventional cryopreservation group.
  • the coated cryopreservation group used the aloe polysaccharide-coated cryopreservation tube prepared in Example 1, and the conventional cryopreservation group directly used the untreated Corning cryopreservation tube. 5 repetitions were set at each time point in each group.
  • Nucleated cell count After diluting the cell suspension with leukocyte diluent, count the hemocytometer, repeat 3 times and take the mean value.
  • Count viable cells by trypan blue exclusion method take 0.5 mL of cell suspension and place it in an EP tube, then add about 0.1 mL of 0.5% trypan blue staining solution, mix for 2 minutes, and then prepare for microscopic examination. The trypan blue rejection rate was calculated using a hemocytometer.
  • Table 1 shows the number of nucleated cells and the trypan blue exclusion rate of amniotic fluid mesenchymal stem cells in the coated cryopreservation group and the conventional cryopreservation group after cryopreservation for 6 months.
  • Table 1 shows that the cryopreservation tube coated with aloe polysaccharide has a cryopreservation effect on amniotic fluid mesenchymal stem cells, and the number of cells and cell viability in the coated cryopreservation group cryopreserved at -80 °C for 6 months are significantly higher than those of conventional cryopreservation Group.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Mechanical Engineering (AREA)
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Abstract

一种含有芦荟多糖包被层的羊水间充质干细胞冻存管，该冻存管主体的内壁设有包被层，包被层为芦荟多糖材质。该冻存管内壁含有芦荟多糖包被层，使用该冻存管冻存羊水间充质干细胞时，-80℃冻存6个月细胞数量和活性无明显下降，冻存保护效果显著优于常规的细胞冻存管。

Description

一种含有芦荟多糖包被层的羊水间充质干细胞冻存管 技术领域
本实用新型属于干细胞领域，涉及干细胞冻存管，具体涉及一种含有芦荟多糖(Pullulan)包被层的羊水间充质干细胞冻存管。
背景技术
近年来，通过对羊水间充质干细胞进行体外细胞培养，研究增殖细胞的生物特性，诱导分化情况，证明了这种细胞具有多向分化的潜能，可以作为种子细胞用于临床方面的研究。
羊水间充质干细胞研究的一个重要方向是如何提高其冻存复苏后的活性。
发明内容
本实用新型的目的在于提供一种含有芦荟多糖包被层的羊水间充质干细胞冻存管，以提高羊水间充质干细胞冻存复苏后的活性。
上述目的是通过如下技术方案实现的：
一种羊水间充质干细胞冻存管，包括冻存管主体，该冻存管主体的内壁设有包被层，包被层为芦荟多糖材质。
进一步地，芦荟多糖包被层的厚度为0.4-0.5mm。
有益效果：
本实用新型提供的冻存管内壁含有芦荟多糖包被层，使用本实用新型冻存管冻存羊水间充质干细胞时，-80℃冻存6个月细胞数量和活性无明显下降，冻存保护效果显著优于常规的细胞冻存管。
附图说明
图1为本实用新型冻存管结构示意图，图中1为芦荟多糖包被层。
具体实施方式
实施例1冻存管的制备
取市售2mL康宁冻存管自制本实用新型提供的冻存管，具体步骤如下：
步骤S1，将芦荟多糖(采用γ射线钴60照射灭菌，购自Shanghai yuanye Bio-Technology Co.,Ltd，货号：B24395)溶于无菌水中，制成浓度为3mg/mL的芦荟多糖溶液，备用；
步骤S2，取2mL上述溶液加入2mL康宁冻存管中，室温静置过夜；
步骤S3，慢慢倾倒冻存管中溶液后，50℃干燥1h，即可在冻存管内壁形成均匀的芦荟多糖包被层，包被层厚度0.4-0.5mm。
实施例2冻存管内壁包被对冻存效果的影响
一、实验方法
1、羊水间充质干细胞培养
将人羊水间充质干细胞用含10％胎牛血清和10ng/mL b-FGF的DMEM/F12培养基于37℃、5％CO 2、饱和湿度条件下培养。
2、冻存保护剂的组成
以含5％DMSO(二甲基亚砜，体积百分浓度)、3％HES(羟乙基淀粉，质量体积百分浓度)和4％HSA(人血白蛋白，质量体积百分浓度)的RPMI 1640培养液为冻存保护剂。
3、低温冻存
取生长状态良好的人羊水间充质干细胞，PBS洗涤，用4℃预冷的冻存保护剂制成细胞悬液，分装于2mL的冻存管中，每管5×10 8个细胞。在4℃冰箱中平衡30min后置于-80℃冰箱直接冻存，冻存6个月后42℃快速复温并检测各项指标。
实验分为包被冻存组和常规冻存组，包被冻存组采用实施例1制备的芦荟多糖包被冻存管，常规冻存组直接使用未处理的康宁冻存管。每组每个时间点设5个重复。
4、指标检测
有核细胞计数：用白细胞稀释液稀释细胞悬液后，血细胞计数板计数，重复3次取均值。
采用台盼蓝拒染法进行活细胞计数：取0.5mL细胞悬液置于EP管，再加入约0.1mL 0.5％台盼蓝染液，混合2min后制片，镜检。采用血细胞计数板计数，计算台盼蓝拒染率。
二、实验结果
包被冻存组和常规冻存组羊水间充质干细胞冻存6个月后有核细胞数和台盼蓝拒染率如表1所示。
表1各指标检测结果(n＝5)
Figure PCTCN2020127852-appb-000001
表1表明，包被有芦荟多糖的冻存管对羊水间充质干细胞具有冻存保护作用，包被冻存组-80℃冻存6个月的细胞数量和细胞活性显著高于常规冻存组。

Claims (2)

  1. 一种羊水间充质干细胞冻存管，包括冻存管主体，其特征在于：该冻存管主体的内壁设有包被层，包被层为芦荟多糖材质。
  2. 根据权利要求1所述的羊水间充质干细胞冻存管，其特征在于：芦荟多糖包被层的厚度为0.4-0.5mm。
PCT/CN2020/127852 2020-11-10 2020-11-10 一种含有芦荟多糖包被层的羊水间充质干细胞冻存管 WO2022099472A1 (zh)

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DE212020000278.9U DE212020000278U1 (de) 2020-11-10 2020-11-10 Kryokonservierungsrohr für mesenchymale Stammzellen im Fruchtwasser mit Überzugsschicht aus Aloe-Polysaccharid

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