Optimization of Slow-Freezing Protocols for Small Intestinal Organoids

Jia Shuqin Cao Yukun Hu Yejing Zhou Xinli

doi:10.12465/issn.0253-4339.20260207002

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Optimization of Slow-Freezing Protocols for Small Intestinal Organoids

更新时间：2026-06-29

- Optimization of Slow-Freezing Protocols for Small Intestinal Organoids
- Journal of Refrigeration Pages: 1-10(2026)
- 作者机构：
  
  上海理工大学健康科学与工程学院生物系统热科学研究所上海 200093
- 作者简介：
  
  Zhou Xinli， female， professor， Institute of Biothermal Science，University of Shanghai for Science and Technology， 86-13817547878， E-mail：zjulily@163.com. Research fields： cryobiomedical technology.
- 基金信息：
- DOI：10.12465/issn.0253-4339.20260207002
  CLC： R318.52;Q813.1
- Received：07 February 2026，
  
  Revised：2026-02-25，
  
  Accepted：20 March 2026，
  
  Online First：29 June 2026，
- 稿件说明：
移动端阅览
贾淑芹，曹裕坤，胡叶静，等. 小肠类器官慢速冷冻工艺的优化研究［J］. 制冷学报，XXXX，XX（XX）：1-10.

Jia Shuqin，Cao Yukun，Hu Yejing，et al. Optimization of Slow-Freezing Protocols for Small Intestinal Organoids［J］. Journal of Refrigeration，XXXX，XX（XX）：1-10.
贾淑芹，曹裕坤，胡叶静，等. 小肠类器官慢速冷冻工艺的优化研究［J］. 制冷学报，XXXX，XX（XX）：1-10. DOI： 10.12465/issn.0253-4339.20260207002.

Jia Shuqin，Cao Yukun，Hu Yejing，et al. Optimization of Slow-Freezing Protocols for Small Intestinal Organoids［J］. Journal of Refrigeration，XXXX，XX（XX）：1-10. DOI： 10.12465/issn.0253-4339.20260207002.

摘要

小肠类器官在肠道生理研究与药物筛选中具有重要价值，但其冷冻保存技术常面临复苏存活率低、结构与功能受损等瓶颈。本文旨在通过引入冷冲击置核技术并优化降温参数，建立一种高效、稳定的小肠类器官慢速冷冻方案。在传统慢速冷冻工艺中引入置核环节，系统研究置核温度、冰晶生长阶段终点温度及降温速率对冻存效果的影响。通过检测复苏后的存活率评估冻存效果，采用H&E染色观察形态完整性、免疫荧光检测增殖标志物及连接蛋白，并利用RT-qPCR定量检测干性指标和多分化谱系的表达水平。置核技术显著提升了复苏效果，存活率从（85.05±3.06）%提升至（88.84±1.78）%，H&E显示置核后结构较完整。-8 ℃置核并以0.5 ℃/min的速度降温至-60 ℃，其存活率（89.02%±2.06%）显著优于传统降温盒组（83.11%±2.45%）。形态学和相关染色进一步证实，优化组类器官复苏后结构致密，增殖能力增强。同时，干性标志物、上皮连接蛋白及多谱系分化相关基因表达均显著上调，成功维持了全谱系分化潜能。置核技术通过主动诱导有序成核，有效降低了过冷度引起的机械损伤。优化后的工艺在存活率、结构完整性及生物学功能维持方面均优于传统方法，为类器官库构建及标准化应用提供了可靠技术方案。

Abstract

Objective

Small-intestinal organoids are highly valuable for intestinal physiological research and drug screening. However， their cryopreservation is often limited by low recovery rates as well as structural and functional damage. This study aimed to establish an efficient and stable slow-freezing protocol for small intestinal organoids by introducing controlled ice nucleation （seeding） and optimizing the key cooling parameters.

Methods

Focusing on slow-freezing， a seeding step was incorporated into the conventional protocol to investigate the effects of seeding temperature， terminal temperature for ice crystal growth， and cooling rate on cryopreservation outcomes. Evaluations included assessing survival rates， H&E staining for morphology， and immunofluorescence for proliferation markers and junction proteins. RT-qPCR was used to measure the expression of stemness and multilineage markers.

Results and Discussions

Seeding significantly enhanced post-thaw recovery， increasing viability from （85.05±3.06）% to （88.84±1.78）%， with H&E staining revealing better organoid architectural preservation. Parameter optimization identified an optimal protocol consisting of ice nucleation at -8 ℃ followed by cooling at 0.5 ℃/min to -60 ℃. This optimized regimen achieved a significantly higher survival rate （89.02%±2.06%） than the conventional passive cooling box method （83.11%±2.45%）. Morphological and immunostaining analyses demonstrated that recovered organoids exhibited compact structures and enhanced proliferation. Additionally， upregulated expression of stemness， epithelial junction integrity， and multilineage genes confirmed the effective preservation of their full-spectrum differentiation potential.

Conclusions

Seeding actively induces ordered ice nucleation， thereby reducing the mechanical damage caused by excessive supercooling. The optimized protocol outperforms traditional methods in survival， structural integrity， and biological function， thus providing technical support for organoid biobanking and standardized applications.

关键词

Keywords

references

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