WO2022099456A1 - 一种含有普鲁兰糖包被层的羊水间充质干细胞冻存管 - Google Patents
一种含有普鲁兰糖包被层的羊水间充质干细胞冻存管 Download PDFInfo
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- WO2022099456A1 WO2022099456A1 PCT/CN2020/127800 CN2020127800W WO2022099456A1 WO 2022099456 A1 WO2022099456 A1 WO 2022099456A1 CN 2020127800 W CN2020127800 W CN 2020127800W WO 2022099456 A1 WO2022099456 A1 WO 2022099456A1
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- mesenchymal stem
- amniotic fluid
- cryopreservation
- stem cell
- fluid mesenchymal
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- 210000004381 amniotic fluid Anatomy 0.000 title claims abstract description 17
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 17
- 229920001218 Pullulan Polymers 0.000 title claims abstract description 16
- 239000004373 Pullulan Substances 0.000 title claims abstract description 16
- 235000019423 pullulan Nutrition 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 38
- 239000011247 coating layer Substances 0.000 claims description 12
- 238000009495 sugar coating Methods 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 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
- 239000000243 solution Substances 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 3
- 239000002577 cryoprotective agent Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 2
- 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
- 238000011084 recovery Methods 0.000 description 2
- 210000000130 stem cell Anatomy 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
- 238000004113 cell culture Methods 0.000 description 1
- 230000003833 cell viability Effects 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
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002062 proliferating effect Effects 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- A01N1/02—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
Definitions
- 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 a pullulan coating layer.
- amniotic fluid mesenchymal stem cell research is how to improve its activity after cryopreservation and recovery.
- the purpose of the utility model is to provide a cryopreservation tube of amniotic fluid mesenchymal stem cells containing a pullulan sugar coating layer, so as to improve the activity of 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 pullulan.
- the thickness of the pullulan sugar coating layer is 0.3-0.5mm.
- the inner wall of the cryopreservation tube provided by the utility model contains a pullulan sugar coating layer.
- Figure 1 is a schematic structural diagram of a cryopreservation tube of the present invention, and Figure 1 is a pullulan sugar coating layer.
- step S1 pullulan (sterilized by ⁇ -ray cobalt 60 irradiation, purchased from Shanghai Yuanye Bio-Technology Co., Ltd, CAS No.: 9057-02-7) was dissolved in sterile water to prepare a concentration of 3 mg /mL of pullulan 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 pullulan coating layer can be formed on the inner wall of the freezing tube, and the coating layer thickness is 0.3-0.5 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 well-grown human amniotic fluid mesenchymal stem cells were taken, washed with PBS, and made into a cell suspension with a cryoprotectant pre-cooled at 4 °C. After equilibrating in a 4°C refrigerator for 30 minutes, it was placed in a -80°C refrigerator for direct freezing. After 6 months of freezing, the cells were quickly rewarmed at 42°C and various indicators were detected.
- the experiment was divided into a coated cryopreservation group and a conventional cryopreservation group.
- the coated cryopreservation group used the pullulan sugar-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 pullulan has a cryopreservation effect on amniotic fluid mesenchymal stem cells, and the cell number and cell viability of the coated cryopreservation group frozen at -80°C for 6 months are significantly higher than those of the conventional cryopreservation group. Cryopreservation group.
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Mechanical Engineering (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
一种含有普鲁兰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，包被层为普鲁兰糖材质。使用所述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，冻存保护效果优于常规的细胞冻存管。
Description
本实用新型属于干细胞领域，涉及干细胞冻存管，具体涉及一种含有普鲁兰糖(Pullulan)包被层的羊水间充质干细胞冻存管。
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普鲁兰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，包被层为普鲁兰糖材质。
进一步地，普鲁兰糖包被层的厚度为0.3-0.5mm。
本实用新型提供的冻存管内壁含有普鲁兰糖包被层，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。
图1为本实用新型冻存管结构示意图，图中1为普鲁兰糖包被层。
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，将普鲁兰糖(采用γ射线钴60照射灭菌，购自Shanghai yuanye Bio-Technology Co.,Ltd，CAS号：9057-02-7)溶于无菌水中，制成浓度为3mg/mL的普鲁兰糖溶液，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普鲁兰糖包被层，包被层厚度0.3-0.5mm。
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制备的普鲁兰糖包被冻存管，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)
表1表明，包被有普鲁兰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)
- 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，包被层为普鲁兰糖材质。
- 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：普鲁兰糖包被层的厚度为0.3-0.5mm。
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/CN2020/127800 WO2022099456A1 (zh) | 2020-11-10 | 2020-11-10 | 一种含有普鲁兰糖包被层的羊水间充质干细胞冻存管 |
| DE212020000279.7U DE212020000279U1 (de) | 2020-11-10 | 2020-11-10 | Kryokonservierungsrohr für mesenchymale Stammzellen im Fruchtwasser mit Überzugsschicht aus Pullulan |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150087056A1 (en) * | 2012-01-17 | 2015-03-26 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Cryopreservation of cells, tissues and organs |
| 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 |
| CN208549786U (zh) * | 2018-06-16 | 2019-03-01 | 上海莱威生物科技有限公司 | 一种用于脐带造血干细胞的冻存管 |
| CN110951681A (zh) * | 2019-12-27 | 2020-04-03 | 南京温博生物科技有限公司 | 一种间充质干细胞培养基 |
| CN111248193A (zh) * | 2020-04-18 | 2020-06-09 | 李刚 | 一种人羊膜间充质干细胞冻存液及其冻存方法 |
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- 2020-11-10 WO PCT/CN2020/127800 patent/WO2022099456A1/zh active Application Filing
- 2020-11-10 DE DE212020000279.7U patent/DE212020000279U1/de active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150087056A1 (en) * | 2012-01-17 | 2015-03-26 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Cryopreservation of cells, tissues and organs |
| 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 |
| CN208549786U (zh) * | 2018-06-16 | 2019-03-01 | 上海莱威生物科技有限公司 | 一种用于脐带造血干细胞的冻存管 |
| CN110951681A (zh) * | 2019-12-27 | 2020-04-03 | 南京温博生物科技有限公司 | 一种间充质干细胞培养基 |
| CN111248193A (zh) * | 2020-04-18 | 2020-06-09 | 李刚 | 一种人羊膜间充质干细胞冻存液及其冻存方法 |
Non-Patent Citations (1)
| Title |
|---|
| WONG VICTOR W., RUSTAD KRISTINE C., GLOTZBACH JASON P., SORKIN MICHAEL, INAYATHULLAH MOHAMMED, MAJOR MELANIE R., LONGAKER MICHAEL : "Pullulan Hydrogels Improve Mesenchymal Stem Cell Delivery into High-Oxidative-Stress Wounds", MACROMOLECULAR BIOSCIENCE, 12 October 2011 (2011-10-12), DE , pages 1458 - 1466, XP055929576, ISSN: 1616-5187, DOI: 10.1002/mabi.201100180 * |
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