CN101250499A - A non-toxic cryopreservation solution for stem cells - Google Patents
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- 231100000252 nontoxic Toxicity 0.000 title claims abstract description 16
- 230000003000 nontoxic effect Effects 0.000 title claims abstract description 16
- 238000005138 cryopreservation Methods 0.000 title abstract description 24
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 32
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 32
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims abstract description 24
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- 239000011550 stock solution Substances 0.000 claims 2
- 230000002528 anti-freeze Effects 0.000 abstract description 16
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- 230000008569 process Effects 0.000 abstract description 7
- 238000001816 cooling Methods 0.000 abstract description 5
- 238000010438 heat treatment Methods 0.000 abstract description 5
- 238000001953 recrystallisation Methods 0.000 abstract description 5
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- 235000013772 propylene glycol Nutrition 0.000 abstract description 4
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 16
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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Abstract
本发明公开了一种干细胞无毒冻存液，其每100毫升溶液中含有：聚乙烯醇0.1～5克，1，2－丙二醇1～30克，余量为水。本发明的冻存液同时含渗透性和非渗透性低温保护剂，尤其采用聚乙烯醇为非渗透性防冻剂组分，具有明显的阻止冰晶生长及抑止重结晶作用，可以有效形成低温玻璃化冻存效果。本发明的玻璃化冻存液体系同时考虑了保护剂的毒性、渗透性、玻璃化能力、玻璃化稳定性以及升温降温速率等因素。所以不只在降温过程中易于进入玻璃化状态，而且还能保证玻璃化溶液在升温过程中的稳定性。本发明的多组分冻存液具有简单而且通用的效果，适于各种细胞、组织及器官的玻璃化冻存。The invention discloses a non-toxic cryopreservation solution for stem cells, which contains 0.1-5 grams of polyvinyl alcohol, 1-30 grams of 1,2-propanediol and water as the balance in every 100 milliliters of the solution. The cryopreservation solution of the present invention contains both permeable and non-permeable low-temperature protective agents, especially polyvinyl alcohol is used as the non-permeable antifreeze component, which has obvious effects of preventing ice crystal growth and recrystallization, and can effectively form low-temperature vitrification Save the effect. The vitrification liquid system of the present invention simultaneously considers factors such as the toxicity, permeability, vitrification ability, vitrification stability and heating and cooling rate of the protective agent. Therefore, it is not only easy to enter the vitrified state during the cooling process, but also can ensure the stability of the vitrified solution during the heating process. The multi-component cryopreservation solution of the present invention has simple and universal effects, and is suitable for vitrification cryopreservation of various cells, tissues and organs.
Description
技术领域 technical field
本发明涉及一种用于保存干细胞的冻存液。The invention relates to a cryopreservation solution for preserving stem cells.
背景技术 Background technique
干细胞移植已成为当今医学领域生物治疗应用研究的热点。为了满足移植的需要，建立干细胞库，长期妥善保存干细胞，已引起研究者们的广泛重视。干细胞的保存方法直接影响细胞的存活率及移植后的效果。目前，临床上唯一可以实现干细胞长期保存的方法就是超低温保存。超低温保存是将生物标本冷冻至-80～-196℃的低温，利用细胞内新陈代谢减慢乃至停止的特性，从而实现较长时间的保存。超低温保存过程具有几个关键性的环节，如：低温保护剂的选择与加入、冷冻方案、保存温度以及复温过程。关于选用何种保护剂、如何降温并保存、以及如何复温等，尚未存在统一的方案，还有待进一步研究。Stem cell transplantation has become a hotspot in the application of biological therapy in the medical field today. In order to meet the needs of transplantation, establishing a stem cell bank and properly preserving stem cells for a long time has attracted extensive attention of researchers. The preservation method of stem cells directly affects the survival rate of cells and the effect after transplantation. At present, the only clinical method that can achieve long-term preservation of stem cells is cryopreservation. Ultra-low temperature preservation is to freeze biological specimens to a low temperature of -80 to -196°C, and use the characteristics of slowing down or even stopping the metabolism in cells to achieve long-term preservation. The cryopreservation process has several key links, such as: selection and addition of cryoprotectant, freezing scheme, storage temperature and rewarming process. Regarding which protective agent to choose, how to cool down and preserve, and how to rewarm, there is no unified plan, and further research is needed.
目前，干细胞的超低温保存主要有两种方法：-196℃程序冷冻液氮保存和-80℃低温冰箱直接保存。前者可以实现长期的安全保存，但是程序降温过程耗时较长并且操作复杂；后者虽操作简单费用低，但不适合长期保存，同时还会受到停电的影响。At present, there are two main methods for cryopreservation of stem cells: -196°C program freezing liquid nitrogen preservation and -80°C cryogenic freezer direct preservation. The former can achieve long-term safe storage, but the program cooling process takes a long time and the operation is complicated; although the latter is easy to operate and low in cost, it is not suitable for long-term storage, and it will also be affected by power outages.
低温保护剂是干细胞低温保存必不可少的添加剂，主要用于抑制细胞内外冰晶的形成，保护细胞免受冷冻损伤。低温保护剂可分为渗透性保护剂和非渗透性保护剂。渗透性保护剂多是一些小分子物质，如二甲基亚砜(DMSO)，可渗透进入细胞内部，抑制细胞内结冰，保护细胞内部结构；非渗透性保护剂，如羟乙基淀粉，主要用于抑制细胞外结冰，保护细胞膜。DMSO是干细胞保存最常用的保护剂，10％DMSO可用于干细胞长期保存，5％DMSO联合6％羟乙基淀粉，也已经成功用于造血干细胞的移植。尽管如此，干细胞对DMSO较为敏感，冷冻前和复温后，应尽可能缩短与DMSO接触的时间。通常情况下，人们的做法是，干细胞加入保护剂后迅速冷冻，复温后直接移植进入患者体内。然而，临床研究表明，DMSO具有很高的毒副作用，通常使接受移植的患者产生恶心、呕吐、腹痛等症状；除此，对心血管、呼吸系统、中枢神经以及肾脏等也会产生副作用。解决这一问题的关键是减少保护剂的毒副作用、提高复温后的细胞活性。因此，使用对人体无毒的低温保护剂长期安全保存干细胞，具有极大的研究价值和应用前景。Cryoprotectant is an essential additive for cryopreservation of stem cells. It is mainly used to inhibit the formation of ice crystals inside and outside cells and protect cells from freezing damage. Cryogenic protectants can be divided into penetrating protectants and non-permeable protectants. Permeable protectants are mostly small molecular substances, such as dimethyl sulfoxide (DMSO), which can penetrate into the interior of cells, inhibit intracellular freezing, and protect the internal structure of cells; non-permeable protectants, such as hydroxyethyl starch, It is mainly used to inhibit extracellular freezing and protect cell membranes. DMSO is the most commonly used protective agent for stem cell preservation. 10% DMSO can be used for long-term stem cell preservation. 5% DMSO combined with 6% hydroxyethyl starch has also been successfully used for hematopoietic stem cell transplantation. Nevertheless, stem cells are sensitive to DMSO, and the time of exposure to DMSO should be as short as possible before freezing and after rewarming. Usually, people's practice is that the stem cells are quickly frozen after being added with a protective agent, and then directly transplanted into the patient's body after rewarming. However, clinical studies have shown that DMSO has high toxicity and side effects, and usually causes symptoms such as nausea, vomiting, and abdominal pain in transplanted patients; in addition, it also has side effects on cardiovascular, respiratory system, central nervous system, and kidneys. The key to solving this problem is to reduce the toxic and side effects of protective agents and improve cell activity after rewarming. Therefore, the long-term safe preservation of stem cells using non-toxic cryoprotectants has great research value and application prospects.
发明内容 Contents of the invention
本发明的目的是克服现有技术存在的不足，提供一种对人体无毒，能长期安全保存干细胞的无毒冻存液。The purpose of the present invention is to overcome the shortcomings of the prior art and provide a non-toxic cryopreservation solution that is non-toxic to the human body and can safely preserve stem cells for a long time.
为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
一种干细胞无毒冻存液，每100毫升溶液中含有：A non-toxic freezing solution for stem cells, each 100ml solution contains:
聚乙烯醇 0.1～5克Polyvinyl alcohol 0.1-5 grams
1，2-丙二醇 1～30克1,2-propanediol 1~30g
余量为水。The balance is water.
其优选的比例是每100毫升溶液含有：Its preferred ratio is that every 100 milliliters of solution contains:
聚乙烯醇 1～5克Polyvinyl alcohol 1-5 grams
1，2-丙二醇 10～20克1,2-propanediol 10-20 grams
余量为水。The balance is water.
本发明采用1，2-丙二醇(PE)为渗透性防冻剂，结合聚乙烯醇(PVA)作为非渗透性保护剂用于低温保存干细胞。The invention adopts 1,2-propanediol (PE) as a permeable antifreeze agent and combines polyvinyl alcohol (PVA) as a non-permeable protective agent for low-temperature preservation of stem cells.
PVA作为非渗透性保护剂，可通过抑制细胞外溶液形成冰晶，维持细胞内外渗透压差，从而达到保护细胞膜的目的。PVA用于低温保存，具有以下优点：As a non-permeable protective agent, PVA can protect the cell membrane by inhibiting the formation of ice crystals in the extracellular solution and maintaining the osmotic pressure difference between the inside and outside of the cell. PVA is used for low temperature preservation and has the following advantages:
1.分子链上含有丰富的羟基，具有很强的水合能力；1. The molecular chain is rich in hydroxyl groups and has strong hydration ability;
2.分子量大，增加少量的PVA，就能使溶液的粘度增大，提高溶液的玻璃化转变温度；2. The molecular weight is large, adding a small amount of PVA can increase the viscosity of the solution and increase the glass transition temperature of the solution;
3.在降温过程中，PVA能够延迟溶液结冰，一旦溶液中形成冰晶，PVA分子会吸附在冰晶表面，抑制其长大和重结晶的发生。3. During the cooling process, PVA can delay the freezing of the solution. Once ice crystals are formed in the solution, PVA molecules will be adsorbed on the surface of the ice crystals, inhibiting their growth and recrystallization.
4.PVA的重结晶抑制活性在复温过程中对于保护细胞膜发挥重要的作用，原因是溶液在升温过程中，只要温度低于熔点，水很容易形成冰晶，或者原有的冰晶很容易长大或者发生重结晶，大冰晶的形成，对细胞膜造成的机械损伤是致命的。因此，PVA的存在，能够阻止大冰晶的形成，降低对细胞膜的破坏，起到保护细胞的作用。4. The recrystallization inhibitory activity of PVA plays an important role in protecting the cell membrane during the rewarming process. The reason is that during the heating process of the solution, as long as the temperature is lower than the melting point, the water is easy to form ice crystals, or the original ice crystals are easy to grow. Or recrystallization occurs, the formation of large ice crystals, and the mechanical damage to the cell membrane is fatal. Therefore, the presence of PVA can prevent the formation of large ice crystals, reduce damage to cell membranes, and protect cells.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1.与已公开的干细胞冻存液相比，本发明的冻存液同时含渗透性和非渗透性低温保护剂，尤其采用聚乙烯醇为非渗透性防冻剂组分，具有明显的阻止冰晶生长及抑止重结晶作用，可以有效形成低温玻璃化冻存效果。1. Compared with the disclosed stem cell cryopreservation solution, the cryopreservation solution of the present invention contains both osmotic and non-permeable cryoprotectants, especially polyvinyl alcohol is used as the non-permeable antifreeze component, which has obvious ability to prevent ice crystals Growth and inhibition of recrystallization can effectively form the effect of low temperature vitrification.
2.本发明的玻璃化冻存液体系同时考虑了保护剂的毒性、渗透性、玻璃化能力、玻璃化稳定性以及升温降温速率等因素。所以不只在降温过程中易于进入玻璃化状态，而且还能保证玻璃化溶液在升温过程中的稳定性。而现有冻存技术中玻璃化溶液从低温保存状态平稳复温到常温状态是较难实现的。2. The vitrification solution system of the present invention simultaneously considers factors such as the toxicity, permeability, vitrification ability, vitrification stability, and heating and cooling rate of the protective agent. Therefore, it is not only easy to enter the vitrified state during the cooling process, but also can ensure the stability of the vitrified solution during the heating process. However, in the existing cryopreservation technology, it is difficult to realize the stable rewarming of the vitrified solution from the low temperature storage state to the normal temperature state.
3.本发明的多组分冻存液具有简单而且通用的效果，适于各种细胞、组织及器官的玻璃化冻存。3. The multi-component cryopreservation solution of the present invention has simple and universal effects, and is suitable for vitrification of various cells, tissues and organs.
4.单独使用渗透性或非渗透性保护剂不能满多方面的技术需求，联合使用两类保护剂已是低温保存技术发展的趋势。本发明的低温保护剂有望在安全保存干细胞的剂量范围内，对人体没有毒性，复温后的干细胞无需去除保护剂，可以直接实施移植。4. The use of permeable or non-permeable protective agents alone cannot meet various technical requirements, and the combined use of the two types of protective agents has become the development trend of cryopreservation technology. The cryoprotectant of the present invention is expected to be within the dose range for safe preservation of stem cells, without toxicity to the human body, and the rewarmed stem cells can be directly transplanted without removing the protectant.
具体实施方式 Detailed ways
实施例1Example 1
将预先制备好的聚乙烯醇0.1g作为非渗透性防冻剂，将1，2-丙二醇30g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 0.1g of pre-prepared polyvinyl alcohol as a non-permeable antifreeze agent, 30g of 1,2-propylene glycol as a permeable antifreeze agent, mix the two components to form a PVA/PE system, add water to the system to make 100ml The solution is a non-toxic cryopreservation solution for stem cells.
实施例2Example 2
将预先制备好的聚乙烯醇1g作为非渗透性防冻剂，将1，2-丙二醇20g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 1g of pre-prepared polyvinyl alcohol as a non-permeable antifreeze, and 20g of 1,2-propylene glycol as a permeable antifreeze. Mix the two components to form a PVA/PE system. Add water to the system to make a 100ml solution , which is a non-toxic cryopreservation solution for stem cells.
实施例3Example 3
将预先制备好的聚乙烯醇2g作为非渗透性防冻剂，将1，2-丙二醇30g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 2g of pre-prepared polyvinyl alcohol as non-permeable antifreeze, 30g of 1,2-propylene glycol as permeable antifreeze, mix the two components to form a PVA/PE system, add water to the system to make a 100ml solution , which is a non-toxic cryopreservation solution for stem cells.
实施例4Example 4
将预先制备好的聚乙烯醇2g作为非渗透性防冻剂，将1，2-丙二醇5g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 2g of pre-prepared polyvinyl alcohol as non-permeable antifreeze, 5g of 1,2-propylene glycol as permeable antifreeze, mix the two components to form a PVA/PE system, add water to the system to make a 100ml solution , which is a non-toxic cryopreservation solution for stem cells.
实施例5Example 5
将预先制备好的聚乙烯醇3g作为非渗透性防冻剂，将1，2-丙二醇30g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 3g of pre-prepared polyvinyl alcohol as non-permeable antifreeze, 30g of 1,2-propylene glycol as permeable antifreeze, mix the two components to form a PVA/PE system, add water to the system to make a 100ml solution , which is a non-toxic cryopreservation solution for stem cells.
实施例6Example 6
将预先制备好的聚乙烯醇3g作为非渗透性防冻剂，将1，2-丙二醇1g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 3g of pre-prepared polyvinyl alcohol as non-permeable antifreeze, and 1g of 1,2-propylene glycol as permeable antifreeze, mix the two components to form a PVA/PE system, add water to the system to make a 100ml solution , which is a non-toxic cryopreservation solution for stem cells.
实施例7Example 7
将预先制备好的聚乙烯醇4g作为非渗透性防冻剂，将1，2-丙二醇20g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 4g of pre-prepared polyvinyl alcohol as non-permeable antifreeze, 20g of 1,2-propylene glycol as permeable antifreeze, mix the two components to form a PVA/PE system, add water to the system to make a 100ml solution , which is a non-toxic cryopreservation solution for stem cells.
实施例8Example 8
将预先制备好的聚乙烯醇5g作为非渗透性防冻剂，将1，2-丙二醇10g作为渗透性防冻剂，两个组分混合即成PVA/PE体系，加入水到该体系配成100ml溶液，即为干细胞无毒冻存液。Use 5g of pre-prepared polyvinyl alcohol as non-permeable antifreeze, 10g of 1,2-propylene glycol as permeable antifreeze, mix the two components to form a PVA/PE system, add water to the system to make a 100ml solution , which is a non-toxic cryopreservation solution for stem cells.
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| CNA2008100273520A CN101250499A (en) | 2008-04-11 | 2008-04-11 | A non-toxic cryopreservation solution for stem cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011147118A1 (en) * | 2010-05-26 | 2011-12-01 | 赛业（广州）生物科技有限公司 | Non-programmed protein-free cell cryopreservation medium |
| CN102273438A (en) * | 2011-08-15 | 2011-12-14 | 上海安集协康生物技术有限公司 | Cryopreservation protection excipient for medicinal cell injection |
| CN110144323A (en) * | 2019-05-29 | 2019-08-20 | 江苏省北科生物科技有限公司 | A kind of settle and separate method of peripheral blood immunocyte |
| WO2020207151A1 (en) * | 2019-04-09 | 2020-10-15 | 北京大学第三医院（北京大学第三临床医学院） | Cryopreservation solution without dmso, preparation method therefor and application thereof |
| WO2020207152A1 (en) * | 2019-04-09 | 2020-10-15 | 中国科学院化学研究所 | Serum-free cryopreservation solution and preparation method and application thereof |
| CN111789108A (en) * | 2019-04-09 | 2020-10-20 | 中国科学院化学研究所 | A kind of cryopreservation liquid and preparation method thereof |
| CN117356559A (en) * | 2023-11-14 | 2024-01-09 | 广州赛业百沐生物科技有限公司 | Rat embryo cryopreservation-resuscitation system matched with rat embryo cryopreservation-resuscitation system and application thereof |
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2008
- 2008-04-11 CN CNA2008100273520A patent/CN101250499A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011147118A1 (en) * | 2010-05-26 | 2011-12-01 | 赛业（广州）生物科技有限公司 | Non-programmed protein-free cell cryopreservation medium |
| CN102273438A (en) * | 2011-08-15 | 2011-12-14 | 上海安集协康生物技术有限公司 | Cryopreservation protection excipient for medicinal cell injection |
| WO2020207151A1 (en) * | 2019-04-09 | 2020-10-15 | 北京大学第三医院（北京大学第三临床医学院） | Cryopreservation solution without dmso, preparation method therefor and application thereof |
| WO2020207152A1 (en) * | 2019-04-09 | 2020-10-15 | 中国科学院化学研究所 | Serum-free cryopreservation solution and preparation method and application thereof |
| CN111789108A (en) * | 2019-04-09 | 2020-10-20 | 中国科学院化学研究所 | A kind of cryopreservation liquid and preparation method thereof |
| CN111789108B (en) * | 2019-04-09 | 2022-03-25 | 中国科学院化学研究所 | Cryopreservation liquid and preparation method thereof |
| CN110144323A (en) * | 2019-05-29 | 2019-08-20 | 江苏省北科生物科技有限公司 | A kind of settle and separate method of peripheral blood immunocyte |
| CN117356559A (en) * | 2023-11-14 | 2024-01-09 | 广州赛业百沐生物科技有限公司 | Rat embryo cryopreservation-resuscitation system matched with rat embryo cryopreservation-resuscitation system and application thereof |
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