WO2022099467A1 - 一种含有多聚半乳糖醛酸包被层的脐带间充质干细胞冻存管 - Google Patents
一种含有多聚半乳糖醛酸包被层的脐带间充质干细胞冻存管 Download PDFInfo
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- WO2022099467A1 WO2022099467A1 PCT/CN2020/127839 CN2020127839W WO2022099467A1 WO 2022099467 A1 WO2022099467 A1 WO 2022099467A1 CN 2020127839 W CN2020127839 W CN 2020127839W WO 2022099467 A1 WO2022099467 A1 WO 2022099467A1
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- umbilical cord
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 18
- 210000003954 umbilical cord Anatomy 0.000 title claims abstract description 18
- 229920002230 Pectic acid Polymers 0.000 title claims abstract description 16
- 239000010318 polygalacturonic acid Substances 0.000 title claims abstract description 16
- 239000011248 coating agent Substances 0.000 title abstract description 5
- 238000000576 coating method Methods 0.000 title abstract description 5
- 238000005138 cryopreservation Methods 0.000 claims description 37
- 239000011247 coating layer Substances 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 3
- 239000002577 cryoprotective agent Substances 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- GUTLYIVDDKVIGB-OUBTZVSYSA-N Cobalt-60 Chemical compound [60Co] GUTLYIVDDKVIGB-OUBTZVSYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 229920001612 Hydroxyethyl starch Polymers 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000005251 gamma ray Effects 0.000 description 1
- 229940050526 hydroxyethylstarch Drugs 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the utility model belongs to the field of stem cells and relates to a stem cell cryopreservation tube, in particular to an umbilical cord mesenchymal stem cell cryopreservation tube containing a polygalacturonic acid coating layer.
- Umbilical cord mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) refer to a kind of multifunctional stem cells existing in the neonatal umbilical cord tissue, which can differentiate into many kinds of tissue cells and have broad clinical application prospects.
- the purpose of the utility model is to provide a cryopreservation tube of umbilical cord mesenchymal stem cells containing a polygalacturonic acid coating layer, so as to improve the activity of the umbilical cord mesenchymal stem cells after cryopreservation and recovery.
- An umbilical cord 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 polygalacturonic acid material.
- the thickness of the polygalacturonic acid coating layer is 0.4-0.6 mm.
- the inner wall of the cryopreservation tube provided by the utility model contains a polygalacturonic acid coating layer.
- Figure 1 is a schematic structural diagram of a cryopreservation tube of the present invention, and Figure 1 is a polygalacturonic acid coating layer.
- step S1 polygalacturonic acid (sterilized by ⁇ -ray cobalt 60 irradiation, purchased from Shanghai Yuanye Bio-Technology Co., Ltd, item number: S11085) was dissolved in sterile water to prepare a 5 mg/mL solution. Polygalacturonic acid solution, for use;
- 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 polygalacturonic acid coating layer can be formed on the inner wall of the freezing tube, and the coating layer thickness is 0.4-0.6 mm.
- Example 2 The effect of the coating on the inner wall of the cryopreservation tube on the effect of cryopreservation
- Human umbilical cord 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 polygalacturonic acid-coated cryopreservation tube prepared in Example 1, and the conventional cryopreservation group directly used untreated Corning cryopreservation tubes. . 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 umbilical cord 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 polygalacturonic acid has a cryopreservation effect on umbilical cord mesenchymal stem cells. in the routine cryopreservation group.
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Abstract
提供了一种含有多聚半乳糖醛酸包被层的脐带间充质干细胞冻存管，冻存管主体的内壁设有包被层，包被层为多聚半乳糖醛酸材质。使用该冻存管冻存脐带间充质干细胞时，-80℃冻存6个月细胞数量和活性无明显下降。
Description
本实用新型属于干细胞领域，涉及干细胞冻存管，具体涉及一种含有多聚半乳糖醛酸包被层的脐带间充质干细胞冻存管。
脐带间充质干细胞(Mesenchymal Stem Cells，MSCs)是指存在于新生儿脐带组织中的一种多功能干细胞，它能分化成许多种组织细胞，具有广阔的临床应用前景。
脐带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多聚半乳糖醛酸包被层的脐带间充质干细胞冻存管，以提高脐带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：
一种脐带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，包被层为多聚半乳糖醛酸材质。
进一步地，多聚半乳糖醛酸包被层的厚度为0.4-0.6mm。
本实用新型提供的冻存管内壁含有多聚半乳糖醛酸包被层，使用本实用新型冻存管冻存脐带间充质干细胞时，-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。
图1为本实用新型冻存管结构示意图，图中1为多聚半乳糖醛酸包被层。
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，将多聚半乳糖醛酸(采用γ射线钴60照射灭菌，购自Shanghai yuanye Bio-Technology Co.,Ltd，货号：S11085)溶于无菌水中，制成浓度为5mg/mL的多聚半乳糖醛酸溶液，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多聚半乳糖醛酸包被层，包被层厚度0.4-0.6mm。
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、脐带间充质干细胞培养
将人脐带间充质干细胞用含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脐带间充质干细胞，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制备的多聚半乳糖醛酸包被冻存管，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脐带间充质干细胞冻存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)
表1表明，包被有多聚半乳糖醛酸的冻存管对脐带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)
- 一种脐带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，包被层为多聚半乳糖醛酸材质。
- 根据权利要求1所述的脐带间充质干细胞冻存管，其特征在于：多聚半乳糖醛酸包被层的厚度为0.4-0.6mm。
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991001636A1 (en) * | 1989-08-11 | 1991-02-21 | Cell Systems Limited | Biological cryopreservation |
| CN1844371A (zh) * | 2005-04-08 | 2006-10-11 | 上海组织工程研究与开发中心 | 冻存胚胎干细胞的复苏方法 |
| CN102625831A (zh) * | 2009-07-20 | 2012-08-01 | 通用医院公司以麻省总医院名义经营 | 提高低温保存细胞生活力的方法和组合物 |
| WO2016200888A1 (en) * | 2015-06-08 | 2016-12-15 | Corning Incorporated | Digestible substrates for cell culture |
| CN106665562A (zh) * | 2017-03-14 | 2017-05-17 | 南京九寿堂医药科技有限公司 | 一种脐血造血干细胞冻存管 |
| CN206744390U (zh) * | 2017-03-14 | 2017-12-15 | 南京九寿堂医药科技有限公司 | 一种脐血造血干细胞冻存管 |
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-
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- 2020-11-10 WO PCT/CN2020/127839 patent/WO2022099467A1/zh active Application Filing
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|---|---|---|---|---|
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| CN1844371A (zh) * | 2005-04-08 | 2006-10-11 | 上海组织工程研究与开发中心 | 冻存胚胎干细胞的复苏方法 |
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