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Article(id=1239215316270641189, tenantId=1146029695717560320, journalId=1238823019242635269, issueId=1239215308985136031, articleNumber=null, orderNo=null, doi=10.12465/j.issn.0253-4339.2025.04.156, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1722700800000, receivedDateStr=2024-08-04, revisedDate=1724860800000, revisedDateStr=2024-08-29, acceptedDate=1727280000000, acceptedDateStr=2024-09-26, onlineDate=1773381554906, onlineDateStr=2026-03-13, pubDate=1755273600000, pubDateStr=2025-08-16, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773381554906, onlineIssueDateStr=2026-03-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773381554906, creator=13701087609, updateTime=1773381554906, updator=13701087609, issue=Issue{id=1239215308985136031, tenantId=1146029695717560320, journalId=1238823019242635269, year='2025', volume='46', issue='4', pageStart='1', pageEnd='170', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773381553169, creator=13701087609, updateTime=1773381893131, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1239216734947824534, tenantId=1146029695717560320, journalId=1238823019242635269, issueId=1239215308985136031, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1239216734947824535, tenantId=1146029695717560320, journalId=1238823019242635269, issueId=1239215308985136031, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=156, endPage=162, ext={EN=ArticleExt(id=1239215317994500151, articleId=1239215316270641189, tenantId=1146029695717560320, journalId=1238823019242635269, language=EN, title=Mechanism and Optimization Strategy of Cryopreservation Damage in Microcarrier-Hepatocyte Complex, columnId=null, journalTitle=Journal of Refrigeration, columnName=null, runingTitle=null, highlight=null, articleAbstract=

The aim of this study is to optimize the cryopreservation scheme for microcarrier hepatocyte complexes used in artificial liver support systems to improve cell survival and adhesion rates. The effects of cryoprotectant concentration, loading temperature, and method on cell viability were evaluated experimentally. It was found that cell toxicity and osmotic damage were reduced significantly, and higher cell survival and adhesion rates were maintained by using two-step loading of 5% volume fraction dimethyl sulfoxide (Me2SO) at 4 ℃. In addition, we investigated the effects of intracellular ice formation and cooling rate on cell viability and attachment. By performing ice seeding at -6 ℃, the intracellular ice damage was effectively reduced, and the adhesion rate of cells after recovery was improved. The experimental results show that a 10% volume fraction of Me2SO and a cooling rate of 1 ℃/min, despite having high toxicity and osmotic damage, have the best freezing effect due to the smallest difference in thermal expansion. This study provides important techniques for the cryopreservation of microcarrier hepatocyte complexes for artificial liver support systems.

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Liu Baolin, male, professor, Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology, 86-13636524955, E-mail: . Research fields: the cryopreservation of cells, tissues and organs, food and drug refrigeration and drying technology.
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为提高细胞的存活率和贴壁率，优化人工肝支持系统中微载体-肝细胞复合体的冻存方案，通过实验评估了不同冷冻保护剂体积分数、加载温度和方式对细胞冻存效果的影响。研究发现，使用体积分数为5%的二甲亚砜（Me2SO）在4 ℃预冷条件下，通过两步加载法，能显著减少细胞毒性和渗透损伤，保持较高的细胞存活率和贴壁率。此外，还研究了降温速率和胞内冰形成对细胞冻存效果的影响，在-6 ℃植冰可以减轻胞内冰的损伤，提高细胞复苏后的贴壁率。在1 ℃/min降温速率下，体积分数为10%的Me2SO尽管存在较高的毒性和渗透损伤，但由于热膨胀差异最小，冻存效果最佳。研究结果可为人工肝支持系统中微载体-肝细胞复合体的冻存提供技术支撑。

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刘宝林，男，教授，上海理工大学生物系统热科学研究所，13636524955，E-mail：。研究方向：细胞、组织和器官的低温保存、食品药品的冷冻干燥技术。
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试剂名称型号纯度生产厂商/品牌
胎牛血清90010898%美国GEMINI公司
胰蛋白酶TE2004Y0.25%天津灏洋（TBD）生物制品有限公司
二甲亚砜（Me2SO）D103277≥99.9%阿拉丁生物科技有限公司
RPMI-1640C11875500无血清美国GIBCO生物制品有限公司
), ArticleFig(id=1239232335753826391, tenantId=1146029695717560320, journalId=1238823019242635269, articleId=1239215316270641189, language=CN, label=表1, caption=材料与试剂, figureFileSmall=null, figureFileBig=null, tableContent=
试剂名称型号纯度生产厂商/品牌
胎牛血清90010898%美国GEMINI公司
胰蛋白酶TE2004Y0.25%天津灏洋（TBD）生物制品有限公司
二甲亚砜（Me2SO）D103277≥99.9%阿拉丁生物科技有限公司
RPMI-1640C11875500无血清美国GIBCO生物制品有限公司
), ArticleFig(id=1239232335854489692, tenantId=1146029695717560320, journalId=1238823019242635269, articleId=1239215316270641189, language=EN, label=Tab.2, caption=Experimental grouping for toxicity testing of protective agents, figureFileSmall=null, figureFileBig=null, tableContent=
加载温度/℃组别保护剂体积分数/%
4A1/B15
A2/B210
A3/B315
20A4/B45
A5/B510
A6/B615
), ArticleFig(id=1239232335942570083, tenantId=1146029695717560320, journalId=1238823019242635269, articleId=1239215316270641189, language=CN, label=表2, caption=保护剂毒性检测实验分组, figureFileSmall=null, figureFileBig=null, tableContent=
加载温度/℃组别保护剂体积分数/%
4A1/B15
A2/B210
A3/B315
20A4/B45
A5/B510
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微载体-肝细胞复合体冻存损伤机制及优化策略
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郝彬江 , 庾曾颖 , 刘宝林
制冷学报 | 2025,46(4): 156-162
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制冷学报 | 2025, 46(4): 156-162
微载体-肝细胞复合体冻存损伤机制及优化策略
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郝彬江, 庾曾颖, 刘宝林
作者信息
  • 上海理工大学生物系统热科学研究所　上海　200093

通讯作者:

刘宝林，男，教授，上海理工大学生物系统热科学研究所，13636524955，E-mail：。研究方向：细胞、组织和器官的低温保存、食品药品的冷冻干燥技术。
Mechanism and Optimization Strategy of Cryopreservation Damage in Microcarrier-Hepatocyte Complex
Binjiang Hao, Zengying Yu, Baolin Liu
Affiliations
  • Institute of Biothermal and Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China
出版时间: 2025-08-16 doi: 10.12465/j.issn.0253-4339.2025.04.156
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摘要
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为提高细胞的存活率和贴壁率，优化人工肝支持系统中微载体-肝细胞复合体的冻存方案，通过实验评估了不同冷冻保护剂体积分数、加载温度和方式对细胞冻存效果的影响。研究发现，使用体积分数为5%的二甲亚砜（Me2SO）在4 ℃预冷条件下，通过两步加载法，能显著减少细胞毒性和渗透损伤，保持较高的细胞存活率和贴壁率。此外，还研究了降温速率和胞内冰形成对细胞冻存效果的影响，在-6 ℃植冰可以减轻胞内冰的损伤，提高细胞复苏后的贴壁率。在1 ℃/min降温速率下，体积分数为10%的Me2SO尽管存在较高的毒性和渗透损伤，但由于热膨胀差异最小，冻存效果最佳。研究结果可为人工肝支持系统中微载体-肝细胞复合体的冻存提供技术支撑。

低温保存  /  肝细胞-微载体复合体  /  冷冻保护剂  /  热膨胀损伤

The aim of this study is to optimize the cryopreservation scheme for microcarrier hepatocyte complexes used in artificial liver support systems to improve cell survival and adhesion rates. The effects of cryoprotectant concentration, loading temperature, and method on cell viability were evaluated experimentally. It was found that cell toxicity and osmotic damage were reduced significantly, and higher cell survival and adhesion rates were maintained by using two-step loading of 5% volume fraction dimethyl sulfoxide (Me2SO) at 4 ℃. In addition, we investigated the effects of intracellular ice formation and cooling rate on cell viability and attachment. By performing ice seeding at -6 ℃, the intracellular ice damage was effectively reduced, and the adhesion rate of cells after recovery was improved. The experimental results show that a 10% volume fraction of Me2SO and a cooling rate of 1 ℃/min, despite having high toxicity and osmotic damage, have the best freezing effect due to the smallest difference in thermal expansion. This study provides important techniques for the cryopreservation of microcarrier hepatocyte complexes for artificial liver support systems.

cryopreservation  /  hepatocyte-microcarrier complex  /  freeze protectants  /  thermal expansion damage
郝彬江, 庾曾颖, 刘宝林. 微载体-肝细胞复合体冻存损伤机制及优化策略. 制冷学报, 2025 , 46 (4) : 156 -162 . DOI: 10.12465/j.issn.0253-4339.2025.04.156
Binjiang Hao, Zengying Yu, Baolin Liu. Mechanism and Optimization Strategy of Cryopreservation Damage in Microcarrier-Hepatocyte Complex[J]. Journal of Refrigeration, 2025 , 46 (4) : 156 -162 . DOI: 10.12465/j.issn.0253-4339.2025.04.156
正文
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20世纪50年代，人工肝支持系统（artificial liver support system，ALSS）作为辅助治疗设施开始逐渐发展，在肝病早中期通过促进肝再生来治愈患者，而在肝病后期，可延长患者生命，换取肝移植等待时间，以期带来生机。
生物型人工肝（biological artificial liver，BAL）是包含优质足量肝细胞、合适的生物材料和生物反应器的人工肝新形式，具有解毒、生物合成和转化等肝细胞的功能。
黄慧等[1]成功将小鼠和人的跨胚层转分化为肝细胞，并证明这种分化而来的肝细胞也具备良好的肝功能，这为大规模获得功能性肝脏细胞提供了可能。
基于微载体的细胞冷冻保存技术通过提供三维生长环境和物理保护，能够提高细胞的存活率和功能性，同时减少冷冻过程中的机械损伤，相较于传统细胞悬液保存方法，更有利于细胞的长期稳定保存和规模化应用[2]。Lu Zekang等[3]制备出三维多孔微载体，其主要材质为壳聚糖，这种微球尺寸规范、孔径均匀，可重建肝细胞生长微环境，黏附在不同微球表面的细胞之间存在相互作用，是一种极好的支架材料选择。Y. Park等[4]用微载体培养系统，将大鼠成年祖细胞扩增并定向分化为类肝细胞，评估细胞功能后证明，微载体培养是干细胞大规模增殖和分化的有效方法。杨波等[5]将大鼠肝细胞接种于Cytodex-3和Cytopore这2种微载体上，证明微载体3D培养方法能够培养出密度高且功能优良的肝细胞。
杨波等[6]对肝细胞复合体的低温保存进行了系列研究，利用Cytodex-3作为细胞支架，发现这种微载体与肝细胞结合后，即便在低温环境下也能维持细胞的活性和功能。实验结果表明，经过2周的低温保存，细胞的存活率和功能指标（如增殖能力和产物分泌）与直接冻存的细胞相比无显著差异，证实了微载体Cytodex-3与细胞结合的复合体低温保存的可行性，但对于冻存损伤的机制未进行明确探讨。
本实验通过改变保护剂体积分数、加载温度和方式等条件，通过测量复合体冻存后的贴壁率、存活率等来评估复合体的冻存效果，优化复合体冻存方案，为人工肝支持系统中微载体-肝细胞复合体的冻存提供技术支持。
1 材料与方法
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1.1 实验材料
实验用HL-7702肝细胞（上海细胞生物研究所），Cytodex-3微载体（Sigma公司，微球直径133~215 μm），其余实验材料如表1所示。
1.2 实验方法
1.2.1 细胞数量检测方法
上述细胞贴壁率、贴壁细胞存活率及总存活率的计算均涉及细胞的具体数量，本研究使用台盼蓝染液和血球计数板进行计数，根据常规操作[7]进行，具体步骤如下：
1）将酶解离心后的细胞重悬，吸取50 μL至1 mL离心管中；
2）吸取50 μL台盼蓝染液加入管中，静置1~2 min；
3）轻柔吹打使溶液混合均匀后，吸取20 μL混合溶液至血球计数板凹槽内；
4）加盖盖玻片，在光学显微镜下观察，调整显微镜视野至凹槽内的刻度线范围内，选择5处进行拍照，具体计数如式（1）所示。
1.2.2 微载体及贴壁细胞的热膨胀系数测量及计算
根据庾曾颖等[8]的操作，以5 s为间隔时间进行拍照，采用图像处理软件Image-Pro Plus对拍摄的微载体或贴壁细胞进行测量计算。
1.3 微载体-肝细胞复合体的制备
肝细胞复合体的制备方法参考杨波等[6,9]，具体步骤为：
1）硅化处理：取20 mL玻片硅化剂加至六孔培养板中，确保每格底部被试剂浸润，浸泡5 min后用重蒸水冲洗，再置于60 ℃电热箱中烘烤过夜，后转移至传递柜中紫外灭菌30 min，取出置于超净台中备用。
2）微载体灭菌：将0.5 g微载体于40 mL无Ga2+和Mg2+的PBS（phosphate buffered saline）缓冲液内充分泡发后，弃上清液，用30 mL、75%体积分数的酒精洗涤微载体微球2次，除去酒精后各加入40 mL酒精孵育过夜。
3）接种准备：先将于酒精中浸泡过夜的微载体清洗干净，即待微载体自然沉降后吸除上清液，用15 mL的PBS冲洗3遍，最后加入37 ℃水浴锅中预热的20 mL完全培养基漂洗1遍，待其沉降后吸除上清液，再加入12 mL完全培养基，将微载体于溶液中充分吹打均匀后转移至硅化处理后的六孔板中，每格加入约2 mL的微载体溶液，后将孔板整体放入CO2培养箱中，使其处于37 ℃、5%体积分数CO2、70%湿度的环境中。
4）细胞接种：将贴壁培养的肝细胞酶解后制成细胞悬液，取出提前备好的装有微载体的六孔板，将细胞以2×105个/mL的密度接种至微载体溶液中，轻柔吹打后放入培养箱中使其生长。
5）相对静止培养：为使细胞不集聚成团沉底，与微球表面充分接触，需要进行相对静止培养操作。在接种完成的前5 h内，每10 min将六孔板取出，在超净台中匀速水平地摇晃1 min，再将培养板放回培养箱中使细胞可以贴微载体表面生长；从第6小时起，每1 h将培养板取出轻柔摇晃1 min；在第12小时后每间隔3 h进行一次1 min摇晃，持续到第18小时时，停止摇晃操作，将培养板置入培养箱中静置培养，后每2 d更换一次培养基。
1.4 冷冻保护剂的毒性和渗透损伤
冷冻保护剂的浓度（本文浓度均指体积分数）对细胞冻存的效果有较大影响，浓度太高的保护剂对细胞有毒性损伤，且Me2SO在常温下的毒性较大，同时还有渗透损伤，当溶液开始冻结，浓度越高的环境会在细胞内外形成更大的渗透压梯度[10-12]，但若保护剂浓度太低，冻结时产生的胞外冰也会对细胞造成损害。在研究热膨胀差异对复合体冻存效果的影响时，首先进行不同浓度冷冻保护剂的细胞毒性检测。
前人的研究[13]已有结论，保护剂的加载方式与加载温度对细胞毒性有影响，所以本文选择不同的加载方式和温度进行保护剂毒性检测，具体分组如表2所示，其中1~3组实验加载温度为4 ℃，4~6组实验加载温度为20 ℃，A组采用一步直接加载保护剂的方法，B组为两步法缓慢加载冷冻保护剂。
将培养好的微载体-肝细胞复合体从六孔板中取出，每组实验取1 mL，按照不同的实验条件加载保护剂，在加载保护剂0、10、20、30 min时，依次取100 μL对复合体进行光学显微观察，并滴入100 μL台盼蓝染液对细胞总数、细胞贴壁率、贴壁细胞存活率和总存活率进行计数测量，贴壁率、贴壁细胞存活率及细胞总存活率的计算如式（2）~式（4）所示。
1.5 胞内冰损伤
胞内冰是细胞冻存损伤的主要因素，在溶液冻结时，有较大的过冷度会造成溶液降温速率急剧增大，产生胞内冰，伤害细胞结构。为研究胞内冰对复合体肝细胞冻存效果的影响，在降温时进行植冰操作。具体的超声波植冰操作参考李维杰等[14]，使用体积分数为10%的Me2SO进行超声波植冰，超声波强度为0.104 1 W/cm2。植冰温度越低，细胞的过冷度越高，由于过冷度是造成细胞死亡的重要因素，应尽量提高植冰温度，减少过冷度，但植冰温度不能无限制提升，必须低于溶液结晶温度。综合过冷度和溶液结晶温度，植冰温度点设置在-6 ℃。植冰可以降低过冷度，并减小结冰时细胞内外温度梯度，避免产生较快的降温速率。在4 ℃时两步缓慢加载保护剂，以1 ℃/min降温至-40 ℃，其中M2组在-6 ℃进行植冰，N2组直接由20 ℃降至-40 ℃。
2 结果与分析
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2.1 保护剂浓度对细胞的毒性和渗透损伤
根据表2的实验分组，实验后得到保护剂体积分数浓度及加载方式对细胞的影响结果，当加载温度为4 ℃而改变其他条件时，肝细胞的贴壁率和总存活率如图1所示。加载方式对细胞的影响在保护剂浓度较大时更为明显。这是因为分步加载有效减小了细胞内外的初始浓度[12]，减小了渗透压梯度，这在较低浓度时效果不明显，当浓度较大时，分步加载因增加了加载时间，对细胞的毒性影响也随之增大。
当在20 ℃加载保护剂时，细胞的存活率和贴壁率如图2所示。同样地，这2个指标均随时间的增加而降低。对于Me2SO这样的渗透性保护剂而言，在常温（20 ℃）下毒性较大，因此，在加载保护剂时，预先将保护剂冷却至4 ℃可有效减轻其毒性。
由上述讨论可知，选择低温4 ℃加载和两步缓慢加载能显著降低保护剂毒性，将这种条件下3种浓度保护剂对细胞的毒性情况进行对比，如图3所示。由图3可以明显看出，Me2SO浓度越高，细胞的存活率和贴壁率越低，但由于慢速冷冻的保护剂整体浓度不算太高，在20 min时存活率与贴壁率也高于40%。
2.2 胞内冰对细胞的损伤
保护剂加载方式一致，均为两步加载，提前在4 ℃下预冷，则当降温速率为1 ℃/min，保护剂为体积分数为10%的Me2SO时，复合体肝细胞的存活率与贴壁率如图4所示，其中M2组在-6 ℃进行植冰操作，N2组直接由20 ℃降至-40 ℃。由图4可知，植冰对提高肝细胞的贴壁率表现出积极的趋势，对细胞的存活率无显著性差异，这可能是对贴壁细胞而言，胞内冰在相邻细胞间的扩散反而减轻了细胞的过度脱水和体积骤变[15-17]，形成一定保护作用。但为了减轻胞内冰对复合体冻存效果的损害，以下的实验仍将进行植冰操作。
2.3 保护剂浓度对冻存效果的影响
在1 ℃/min降温速率下，微载体和细胞的热膨胀系数变化，与保护剂浓度有关联。要对比两者热膨胀差异最小的实验条件，可将两者的热膨胀变化曲线直接在α-T图中进行对比。图5所示为两者热膨胀系数差异对比，蓝色代表微载体的热膨胀系数，红色为细胞热膨胀系数，其中又分为不同浓度的保护剂环境。其中在10%体积分数的Me2SO环境下的细胞与微载体曲线呈现出最为理想的重合度，红蓝2类曲线更加接近，平均热膨胀差异为（5.22±4.46）×10-4。
当环境温度降至溶液冻结温度（-18~-16 ℃）附近，热膨胀系数出现较大增长，这可以解释为溶液出现部分冻结，溶液内生成冰晶，渗透压增大，微载体内部水分向外迁移，这无疑会引起微载体继续收缩，降至冻结温度（约为-18 ℃）后，热膨胀系数达到峰值，则此时微载体的热膨胀系数由微载体的材质与溶液的渗透压共同影响，实质上是广义的热膨胀系数。
选择实验条件为1 ℃/min的降温速率、-40 ℃的终温，研究不同保护剂浓度下细胞的冻存效果，如图6所示。C1、D1、F1分别对应体积分数为5%、10%、15%的Me2SO保护剂。D1组是1 ℃/min降温速率下微载体与肝细胞热膨胀差异最小的条件，由图6可知，D1组复合体的总存活率和肝细胞的贴壁率均为最高，贴壁率达到40%，总存活率为62.65%± 0.64%。F1组的热膨胀差异最大，而且当保护剂浓度最高时，相应的渗透损伤也最大，其总存活率为47.74%±0.72%，贴壁率为32.7%±0.53%，冻存效果为3组中最差的。
保护剂毒性和渗透压损伤主要通过保护剂浓度的差异来评估，10%体积分数的Me2SO环境下，细胞所受毒性和渗透损伤大于5%体积分数时，但对于1 ℃/min的降温速率而言，10%体积分数时对应的是最小热膨胀系数，冻存后的结果表明，此时细胞的存活率和贴壁率反而更高。
综上所述，在复合体的慢速冻存中，细胞所受的低温损伤主要为保护剂毒性、渗透损伤和热膨胀损伤，将两者综合评估后，可知热膨胀损伤对细胞贴壁率的影响更显著。
2.4 肝细胞复合体在慢速冻存中的损伤类型及损伤机制
图7所示为肝细胞复合体在降温速率为1 ℃/min，保护剂为10%体积分数的Me2SO时的冻存结果。
由图7可知，在温度降至冻结温度（约-18 ℃）之前，贴壁细胞存活率在85%以上，当温度为-18 ℃时，细胞总存活率为70.79%±0.47%，贴壁率和存活率的下降说明这期间细胞受到损伤，此时主要是由于热膨胀差异引起的。温度下降时，微载体和细胞的热膨胀系数也会相应改变，由图5可知，此时两者差异范围在（5.22±4.46）×10-4内，存在一些黏附细胞从微载体上脱落的情况。温度降至冻结温度以下时，细胞的贴壁率骤降，这也反映出冻结时胞外冰对细胞黏附态的损伤。当温度降至-26 ℃，受溶液渗透损伤和热膨胀损伤影响，总存活率降至约64.475%± 1.44%，贴壁率降至43.2%±2.13%。已有研究对慢速降温过程中细胞损伤机制进行了定量分析[18]，认为在刚达到冻结温度时，细胞外冰晶生长引起的机械损伤是损害细胞的主要原因[19]，随温度降低，多数细胞是由于细胞内外溶液渗透差引起的渗透压损伤造成的。细胞热膨胀系数的测量，实质上在到达溶液冻结温度后，贴壁细胞的面积变化不仅是热膨胀变化的结果，细胞内外溶液的渗透压同样也会改变细胞贴壁面积，因此，溶液冻结温度后的热膨胀系数事实上是更广义的概念，“广义热膨胀系数”的变化也受渗透压的影响。
3 结论
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为控制降温过程中胞内冰为复合体冻存带来的损伤，通过在-6 ℃时进行植冰操作，研究胞内冰对复合体冻存的影响。同时，复合体冻存中的保护剂毒性和渗透损伤，通过控制保护剂浓度和保护剂加载方式来衡量，选用对细胞损害最小的方式来加载保护剂。将微载体、肝细胞的热膨胀系数差异导致的热膨胀损伤作为影响复合体的冻存效果的重要因素进行研究，得到结论如下：
1）保护剂浓度的变化对复合体肝细胞的毒性和渗透损伤有显著影响，浓度最小即5%体积分数的Me2SO时，对应的损伤最小，通过4 ℃预冷和两步加载的方法，能使细胞的存活率和贴壁率在第20分钟时仍保持较高的状态。
2）研究保护剂浓度主导的热膨胀差异时，选择降温速率为1 ℃/min降至-40 ℃，对比3种不同保护剂浓度下细胞的贴壁率和存活率，发现10%体积分数的Me2SO时热膨胀差异最小，冻存效果也最好。考虑到保护剂浓度越高，细胞在冻存、复苏后受到的毒性和渗透损伤都越大的情况下，5%体积分数的Me2SO环境下复合体的冻存效果仍比10%体积分数的Me2SO组的效果差，说明热膨胀损伤是影响复合体慢速冻存的巨大因素，且对细胞冻存效果的影响程度大于保护剂的毒性和渗透损伤。
3）研究了降温过程中微载体-肝细胞复合体的冻存曲线随终温变化的规律，重点在于溶液冻结温度前后，细胞存活率和贴壁率的骤降反映了溶液中冰晶生长对贴壁细胞黏附态的破坏。在溶液冻结温度前，热膨胀损伤是影响冻存效果的主要原因；发生冻结瞬间，胞外冰的损伤突起；持续降温后，微载体的热膨胀系数发生较大变化，即此时这种由溶液渗透压改变和热膨胀系数变化同时引起的“广义热膨胀损伤”成为损害细胞的主要因素。
基金
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  • 国家自然科学基金(51776130)
文献
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2025年第46卷第4期
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doi: 10.12465/j.issn.0253-4339.2025.04.156
  • 接收时间：2024-08-04
  • 首发时间：2026-03-13
  • 出版时间：2025-08-16
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  • 收稿日期：2024-08-04
  • 修回日期：2024-08-29
  • 录用日期：2024-09-26
基金
National Natural Science Foundation of China(51776130)
国家自然科学基金(51776130)
作者信息
    上海理工大学生物系统热科学研究所　上海　200093

通讯作者:

刘宝林，男，教授，上海理工大学生物系统热科学研究所，13636524955，E-mail：。研究方向：细胞、组织和器官的低温保存、食品药品的冷冻干燥技术。
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https://castjournals.cast.org.cn/joweb/zlxb/CN/10.12465/j.issn.0253-4339.2025.04.156
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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