AbstractCryopreservation of human corneal stroma-derived mesenchymal stromal cells (hCS-MSCs) with dimethylsulfoxide (DMSO) as a cryoprotective agent (CPA) has not been previously compared to that with glycerol under standard conditions. The hCS-MSCs were hereby cryopreserved with both compounds using a freezing rate of 1 °C/minute.

摘要在标准条件下，用二甲基亚砜（DMSO）作为冷冻保护剂（CPA）冷冻保存人角膜基质来源的间充质基质细胞（hCS-MSCs）之前尚未与甘油进行比较。因此，使用1℃/分钟的冷冻速率将hCS-MSC与两种化合物一起冷冻保存。

The CPAs were tested by different concentrations in complete Minimum Essential Medium (MEM) approved for good manufacturing practice, and a medium frequently used in cell laboratory culturing—Dulbecco’s modified eagle serum. The hCS-MSCs were isolated from cadaveric human corneas obtained from the Norwegian Eye Bank, and immunophenotypically characterized by flow cytometry before and after cryopreservation.

CPA在批准用于良好生产规范的完全最低必需培养基（MEM）和细胞实验室培养Dulbecco改良eagle血清中经常使用的培养基中以不同浓度进行测试。从挪威眼库获得的尸体角膜中分离hCS-MSCs，并在冷冻保存前后通过流式细胞术进行免疫表型表征。

The survival rate, the cellular adhesion, proliferation and cell surface coverage after cryopreservation of hCS-MSCs has been studied. The hCS-MSCs were immunofluorescent stained and examined for their morphology microscopically. The results showed that cryopreservation of hCS-MSCs in MEM with 10% glycerol gives a higher proliferation rate compared to other cryopreserving media tested.

研究了hCS-MSCs冷冻保存后的存活率，细胞粘附，增殖和细胞表面覆盖率。对hCS-MSC进行免疫荧光染色并在显微镜下检查其形态。结果表明，与其他测试的冷冻保存介质相比，在含有10%甘油的MEM中冷冻保存hCS-MSCs具有更高的增殖率。

Based on the results, hCS-MSCs can safely be cryopreserved using glycerol instead of the traditional use of DMSO..

根据结果，hCS-MSCs可以使用甘油而不是传统的DMSO进行冷冻保存。。

IntroductionIn developing countries in Asia and Africa, corneal diseases requiring transplantation often stem from infectious and nutritional issues, while developed countries face inherited degenerative corneal disorders like keratoconus and Fuchs` corneal endothelial dystrophy that may require corneal transplantation or keratoplasty1,2, which is limited by donor scarcity and pre- and post- operative complications2.

引言在亚洲和非洲的发展中国家，需要移植的角膜疾病通常源于感染和营养问题，而发达国家则面临遗传性退行性角膜疾病，如圆锥角膜和Fuchs角膜内皮营养不良，可能需要角膜移植或角膜成形术1,2，这受到供体稀缺和手术前后并发症的限制2。

Taking this into consideration and the millions of patients waiting to be treated for corneal diseases, there is a need for alternatives using regenerative medicine technologies3,4,5.Researchers are exploring cell-based therapy in different medical fields, including ocular diseases3,4,5. To succeed with such therapy, there are certain required traits for the specific cell lines6.

考虑到这一点以及数百万等待治疗角膜疾病的患者，需要使用再生医学技术替代3,4,5。研究人员正在探索不同医学领域的细胞疗法，包括眼部疾病3,4,5。为了成功进行这种治疗，特定细胞系有某些必需的特征6。

Mesenchymal stromal/stem cells (MSCs) have been shown to possess several unique properties (e.g. multilineage differentiation potential, induction of tissue repairment, immunomodulation) for cell-based therapy6. Since 2005, some authors including our group have studied the multipotency of human corneal stroma-derived MSCs (hCS-MSCs)7,8,9,10,11.

间充质基质/干细胞（MSCs）已被证明具有几种独特的特性（例如多向分化潜能，组织修复诱导，免疫调节）用于基于细胞的治疗6。。

These cells are abundant in the central part of the corneal stroma and possess ability to reduce corneal scarring in a mouse model7,10,11, as well as wound healing properties in vitro7.Challenges such as the lack of suitable preservation conditions in good manufacturing practices (GMP) persist12,13.

这些细胞在角膜基质的中央部分丰富，并且具有减少小鼠模型中角膜瘢痕形成的能力7,10,11，以及体外伤口愈合特性7。诸如在良好生产规范（GMP）中缺乏合适的保存条件等挑战持续存在12,13。

Many authors employed basal growth media containing multiple nutrients, growth factors, and fetal bovine serum (FBS), which has been the gold standard medium supplement in many laboratories throughout the world12. However, the FBS can potentially represent a source of zoonotic infections and xenogenic antigens, both of which can impair the cell c.

许多作者使用含有多种营养素，生长因子和胎牛血清（FBS）的基础生长培养基，这是世界各地许多实验室的金标准培养基补充剂12。然而，FBS可能代表人畜共患感染和异种抗原的来源，这两者都可能损害细胞c。

Table 1 Different cell culturing media and cryoprotective agents (CPAs) used in the experiments.Full size tableThree primary hCS-MSCs donors were counted with a Bürker chamber using Tryphan Blue to visualize the live cells and, subsequently, approximately 7.5·105–1·106 cells/mL were cryopreserved in media containing DMSO or glycerol.

表1实验中使用的不同细胞培养基和冷冻保护剂（CPA）。全尺寸表使用台盼蓝用Bürker室对三个原代hCS-MSCs供体进行计数，以可视化活细胞，随后将约7.5·105-1·106个细胞/mL冷冻保存在含有DMSO或甘油的培养基中。

The hCS-MSCs cryopreserved in DMSO were gently mixed and immediately transferred into − 80 °C inside a freezing container (Mr. Frosty; 1 °C/min cooling rate), while the hCS-MSCs cryopreserved with glycerol were gently mixed in the cryovial and left on the bench for 20 min prior to transferring into − 80 °C inside the Mr.

将在DMSO中冷冻保存的hCS-MSCs轻轻混合，并立即转移到冷冻容器内的-80°C（Frosty先生；1°C/min冷却速率），而将用甘油冷冻保存的hCS-MSCs轻轻混合在冷冻瓶中并在台上放置20分钟，然后转移到Mr内的-80°C。

Frosty. All the cryovials were transferred after a few days into liquid nitrogen for at least two weeks storage.Cell surface marker phenotype characterization of the hCS-MSCsThe immunophenotype characterization of the hCS-MSCs was carried out using a BD Accuri™ C6 personal flow cytometer (Becton Dickinson, NJ, USA).

霜冻。几天后，将所有冷冻瓶转移到液氮中保存至少两周。hCS-MSC的细胞表面标记表型表征使用BD Accuri™C6个人流式细胞仪（Becton Dickinson，NJ，USA）进行hCS-MSC的免疫表型表征。

FITC, PE and APC- conjugated antibodies (Biolegend, CA, USA) were obtained, against cell surface- expressed proteins: CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD117, CD146, CD166, CD184 (Sup. Table 1).Briefly, 1·105 dissociated cells were pipetted into individual 1.5 mL tubes (Corning® Axygen®, Merck/Sigma-Aldrich, MO, USA), rinsed and pelleted in cold fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin (Thermo Fisher Scientific, MA, USA) in DPBS (1X)).

获得了针对细胞表面表达蛋白的FITC，PE和APC偶联抗体（Biolegend，CA，USA）：CD31，CD34，CD44，CD45，CD73，CD90，CD105，CD106，CD117，CD146，CD166，CD184（表1）。简而言之，将1.105个解离的细胞移液到单独的1.5 mL管（Corning®Axygen®，Merck/Sigma-Aldrich，MO，USA）中，在冷荧光激活细胞分选（FACS）缓冲液（0.5%牛血清白蛋白（Thermo Fisher Scientific，MA，USA）中冲洗并沉淀在DPBS（1X）中）。

Antibody solutions (1:40) were added to the individual tubes with the cells, gently resuspended and incubated on ice for 30 min. Once completed, the hCS-MSCs were washed twice in cold FACS buffer, centrifuged (3 min, 500 RCF g, 4 °C) then resuspended in 0.2 mL buffer before measurement. The immunophenotype characterization was pe.

将抗体溶液（1:40）与细胞一起加入到各个试管中，轻轻重悬并在冰上孵育30分钟。完成后，将hCS-MSC在冷FACS缓冲液中洗涤两次，离心（3分钟，500 RCF g，4°C），然后在测量前重悬于0.2 mL缓冲液中。。

Data availability

数据可用性

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

研究中提出的原始贡献包含在文章/补充材料中；进一步的询问可以直接联系通讯作者。

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Download referencesAcknowledgementsThe authors are grateful to Hans Christian Aass, Department of Medicine, University of Oslo, Norway, for assistance with flow cytometry. This work was supported by the Norwegian Research Council grant No. E667269 (3D-CORNEA) within the NANO2021 activity.

下载参考文献致谢作者感谢挪威奥斯陆大学医学系的Hans Christian Aass对流式细胞术的帮助。这项工作得到了挪威研究委员会批准号E667269（3D-CORNEA）在NANO2021活动中的支持。

The authors acknowledge also the financial support provided by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 801133. The Center for Eye Research and participants in this project have received funding from the Norwegian Association of the Blind and Partially Sighted and the Raagholt foundation.Author informationAuthors and AffiliationsCenter for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, NorwayKristoffer Larsen, Goran Petrovski & Gerard Boix-LemoncheDepartment of Ophthalmology, Oslo University Hospital, Oslo, NorwayGoran PetrovskiSchool of Medicine, University of Split, 21000, Split, CroatiaGoran PetrovskiUKLONetwork, University St.

作者还感谢欧盟地平线2020研究与创新计划根据第801133号玛丽·斯科多夫斯卡·居里赠款协议提供的财政支持。眼科研究中心和该项目的参与者得到了挪威盲人和部分视力协会和拉格霍尔特基金会的资助。作者信息作者和附属机构奥斯陆大学医学院临床医学研究所眼科研究与创新诊断学系，奥斯陆，NorwayKristoffer Larsen，Goran Petrovski＆Gerard Boix LemoncheDepartment of Ophthalmology，奥斯陆大学医院，奥斯陆，NorwayGoran PetrovskiSchool of Medicine，University of Split，21000，Split，CroatiaGoran PetrovskiUKLONetwork，University St。

Kliment Ohridski –Bitola, 7000, Bitola, North MacedoniaGoran PetrovskiAuthorsKristoffer LarsenView author publicationsYou can also search for this author in.

Kliment Ohridski–Bitola，7000，Bitola，北马其顿共和国彼得罗夫斯基作家克里斯托弗·拉尔森维（KristoferLarsenview）作者出版物您也可以在中搜索这位作者。

PubMed Google ScholarGoran PetrovskiView author publicationsYou can also search for this author in

PubMed Google ScholarGoran Petrovsiview作者出版物您也可以在

PubMed Google ScholarGerard Boix-LemoncheView author publicationsYou can also search for this author in

PubMed Google ScholarGerard Boix LemoncheView作者出版物您也可以在

PubMed Google ScholarContributionsConceptualization, KL, and GB-L; formal analysis, KL, and GB-L; funding acquisition, GP, and GB-L; investigation, KL, and GB-L; methodology, GB-L; supervision, GB-L; writing—review and editing, KL, GP, and GB-L. All authors contributed to the article and approved the submitted version.Corresponding authorCorrespondence to.

PubMed谷歌学术贡献概念化，KL和GB-L；形式分析，KL和GB-L；资金收购，GP和GB-L；调查，KL和GB-L；方法学，GB-L；监督，GB-L；撰写评论和编辑，KL，GP和GB-L。所有作者都为文章做出了贡献并批准了提交的版本。对应作者对应。

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Reprints and permissionsAbout this articleCite this articleLarsen, K., Petrovski, G. & Boix-Lemonche, G. Alternative cryoprotective agent for corneal stroma-derived mesenchymal stromal cells for clinical applications.

转载和许可本文引用本文Larsen，K.，Petrovski，G。＆Boix-Lemonche，G。用于临床应用的角膜基质衍生的间充质基质细胞的替代冷冻保护剂。

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