{"title":"重新发现作为间充质干细胞低温保护剂的聚乙二醇。","authors":"Madhumita Patel, Jin Kyung Park, Byeongmoon Jeong","doi":"10.1186/s40824-023-00356-z","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at -196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required.</p><p><strong>Method: </strong>As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at -196 °C for 7 days. Then, cell recovery was assayed.</p><p><strong>Results: </strong>We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants.</p><p><strong>Conclusions: </strong>PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system.</p>","PeriodicalId":9079,"journal":{"name":"Biomaterials Research","volume":null,"pages":null},"PeriodicalIF":11.3000,"publicationDate":"2023-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9942331/pdf/","citationCount":"0","resultStr":"{\"title\":\"Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells.\",\"authors\":\"Madhumita Patel, Jin Kyung Park, Byeongmoon Jeong\",\"doi\":\"10.1186/s40824-023-00356-z\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at -196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required.</p><p><strong>Method: </strong>As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at -196 °C for 7 days. Then, cell recovery was assayed.</p><p><strong>Results: </strong>We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants.</p><p><strong>Conclusions: </strong>PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system.</p>\",\"PeriodicalId\":9079,\"journal\":{\"name\":\"Biomaterials Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":11.3000,\"publicationDate\":\"2023-02-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9942331/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biomaterials Research\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1186/s40824-023-00356-z\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"Medicine\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biomaterials Research","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1186/s40824-023-00356-z","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Medicine","Score":null,"Total":0}
引用次数: 0
摘要
背景:含二甲基亚砜(DMSO)(10% v/v)的培养基被广泛用于-196 °C的细胞冷冻保存。然而,残留的二甲基亚砜因其毒性而一直引起人们的关注;因此,需要彻底清除二甲基亚砜:方法:作为经美国食品和药物管理局批准用于人类各种生物医学应用的生物相容性聚合物,不同分子量(400、600、1 K、1.5 K、5 K、10 K 和 20 K Da)的聚乙二醇(PEGs)被研究用作间充质干细胞(MSCs)的冷冻保护剂。考虑到不同分子量的 PEG 对细胞的渗透性不同,在-196 °C冷冻保存 7 天之前,将细胞在 10 wt.% 的 PEG 存在下于 37 °C预孵育 0 小时(不孵育)、2 小时和 4 小时。然后检测细胞的恢复情况:结果:我们发现低分子量 PEG(400 和 600 Da)在预孵育 2 小时后表现出卓越的冷冻保护特性,而中分子量 PEG(1 K、1.5 K 和 5 K Da)无需预孵育即可表现出冷冻保护特性。高分子量聚乙二醇(10 K 和 20 K Da)不能作为间充质干细胞的低温保护剂。对 PEG 的冰再结晶抑制(IRI)、冰成核抑制(INI)、膜稳定性和细胞内转运的研究表明,低分子量 PEG(400 和 600 Da)具有良好的细胞内转运特性,因此预孵育期间内化的 PEG 有助于低温保护。中等分子量的 PEG(1 K、1.5 K 和 5 K Da)通过 IRI、INI 以及部分内化的 PEG 发挥细胞外 PEG 的作用。高分子量 PEG(10 K 和 20 K Da)会在预孵育过程中杀死细胞,不能作为低温保护剂:结论:PEG 可用作低温保护剂。结论:PEGs 可用作低温保护剂,但具体操作(包括预孵育)应考虑 PEGs 分子量的影响。回收的细胞增殖良好,并进行了成骨/软骨/成脂分化,与传统的 DMSO 10% 系统回收的间充质干细胞相似。
Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells.
Background: A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at -196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required.
Method: As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at -196 °C for 7 days. Then, cell recovery was assayed.
Results: We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants.
Conclusions: PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system.
期刊介绍:
Biomaterials Research, the official journal of the Korean Society for Biomaterials, is an open-access interdisciplinary publication that focuses on all aspects of biomaterials research. The journal covers a wide range of topics including novel biomaterials, advanced techniques for biomaterial synthesis and fabrication, and their application in biomedical fields. Specific areas of interest include functional biomaterials, drug and gene delivery systems, tissue engineering, nanomedicine, nano/micro-biotechnology, bio-imaging, regenerative medicine, medical devices, 3D printing, and stem cell research. By exploring these research areas, Biomaterials Research aims to provide valuable insights and promote advancements in the biomaterials field.