高效分离猪脂肪组织中脂肪来源的干细胞和脂肪细胞
2026年5月26日
本文介绍了一种高效分离脂肪来源干细胞和脂肪细胞的标准方法，其特点是处理时间缩短>90%，细胞存活率高且兼容性广泛。
本研究旨在高效且快速地从脂肪组织中分离细胞。传统消化需要耗时，而且可能会过度消化。我们的酶波和机械波长测量提供了温和的扩散，并缩短了隔离时间。
首先，将一块安乐死猪的皮下脂肪组织放入培养皿中。沿脂肪组织与真皮之间的自然平面进行解剖，获得一整张厚度为2至3毫米的ULB薄片。用冰冷无菌生理盐水冲洗一次ULB。
撕开无菌培养皿后，用预先消毒的天平称重无菌皿中的组织。每取1.8克组织，加入10毫升冰冷的D-Hank's缓冲液，并保持组织完全浸没。使用无菌手术剪刀识别并切除组织中所有可见的血管。
去除所有结缔组织和纤维化成分。将切除的材料丢弃到指定的生物危害容器中。冲洗D-Hank缓冲液中的组织，去除残留的血液和杂质。
撕开培养皿后，用预先灭菌的天平称重无菌培养皿中的组织。将1.8克冲洗组织碎片转移到无菌的1.5毫升微型离心管中。向管子中加入800微升A型组织解离缓冲液。
使用无菌眼科学剪将管内组织切碎，大小约为一到两立方毫米。将绞碎组织连同周围缓冲液转移到新的微型离心管。向管子中加入200微升A型组织解离剂。
轻轻旋转并用手摇晃管子三次，搅拌成分。确保脂肪碎片完全浸入解离溶液中。检查管盖是否紧闭。
将管子放入37摄氏度的金属浴中，孵育5到10分钟。将部分消化的脂肪组织在缓冲液中转移到专用于机械解离系统的无菌1.5毫升微型离心管中。运行预设的脂肪组织程序，使用200赫兹正弦波进行机械解离。
收集细胞悬浮液，通过200微米的细胞筛，放入新的50毫升离心管中。在滤网中加入5毫升预冷的D-Hank溶液，温度为4摄氏度。用D-Hank溶液冲洗滤网两次。
将过滤后的细胞悬浮液以500克离心，温度为4摄氏度，离心五分钟。使用无菌巴斯德移液器抽取并丢弃上清液脂质层，同时保留一毫升缓冲液保护脂肪细胞层。采集含有脂肪细胞的第二层，同时不扰动第三和第四层。
吸入并丢弃第三层。向底部含有气质血管分数（SVF）的沉淀加入一毫升D.Hank缓冲液。轻柔地将细胞沉淀重新悬浮于缓冲液中，达到细胞浓度为2到5乘以10的6细胞每毫升的幂次方。
将SVF电池悬浮液置于1.5毫升微型离心管中。使用前保持悬挂冰冻状态。在无菌0.5毫升微型离心管中加入10微升SVF细胞悬浮液和10微升0.4%的锥蓝溶液。
轻轻上下移液管三次。将10微升染色细胞混合物转移到适合自动细胞计数器的计数载玻片上。确认样品完全充满腔室且不溢出。
将计数滑片插入自动单元计数器。选择锥蓝测定法类型，必要时调整细胞浓度范围。点击计数键即可启动自动细胞计数。
监测计数室图像，观察活细胞和死细胞的自动区分。记录细胞存活率百分比及仪器屏幕上显示的活细胞总数。对每个样本进行三次独立测量。
计算每克脂肪组织的平均存活率和细胞产率。在D.Hank's中制备5微克/毫升的染色液和1微摩尔BODIPY。将分离的脂肪细胞在染色溶液中以37摄氏度孵育15分钟。
使用自制的适合脂肪细胞的载玻片盖玻璃系统，用于适用于荧光显微镜的载玻片准备。自制的适合脂肪细胞的安装系统实现了完整脂肪细胞均匀分布，以便清晰成像。与传统方法产生拥挤多层排列不同，提取的脂肪细胞结构完整，形态正常。
机械解离法获得的脂肪细胞存活率显著高于传统酶水解法。纯化培养的脂肪衍生干细胞表现出均匀的成纤维细胞形态，细胞呈细长的纺锤形，排列有序。大多数细胞保持未染色，表明存活性，而只有少数细胞呈蓝色，代表死亡细胞。
该方案使研究人员能够比较脂肪库中分离的细胞特征。该方案中分离的SVF可用于类器官构建和脂肪干细胞纯化。未来研究有望进一步优化分离方法，考虑不同仓库中细胞外基质脂肪组织的差异。
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Overview
This article presents a rapid and efficient protocol for isolating adipose-derived stem cells (ADSCs) from adipose tissue using a combination of enzymatic digestion and mechanical wave-based dissociation. The method, exemplified by the SoniConvert system, significantly reduces processing time, improves cell viability, and yields high-quality single-cell suspensions suitable for downstream applications in regenerative medicine and research.
Key Study Components
Area of Science
- Regenerative medicine
- Cell biology
- Stem cell isolation
Background
- ADSCs are valuable for tissue repair, disease modeling, and cell therapy due to their multipotency and immunomodulatory properties.
- Traditional isolation methods are time-consuming (1–3 hours), yield low cell viability (<70%), and suffer from batch variability.
- Efficient, reproducible isolation of high-viability ADSCs is critical for research and clinical translation.
- Mechanical wave-based dissociation offers a potential solution to these limitations.
Purpose of Study
- To develop and demonstrate a rapid, gentle, and reproducible protocol for isolating ADSCs from adipose tissue.
- To compare the new method's efficiency and cell viability with traditional enzymatic digestion protocols.
- To provide a platform compatible with various downstream applications, including organoid construction and cell culture.
Methods Used
- Harvesting and preparation of subcutaneous adipose tissue from euthanized pigs.
- Enzymatic digestion using a tissue-specific dissociation buffer and agent.
- Mechanical dissociation via a preset program using a 200 Hz sinusoidal wave (SoniConvert system).
- Filtration, centrifugation, and separation of stromal vascular fraction (SVF) and adipocytes.
- Cell viability assessment using trypan blue staining and automated cell counting.
- Fluorescence staining and microscopy to assess adipocyte morphology and integrity.
Main Results
- The core isolation process is completed in approximately 30 minutes, representing a >90% reduction in processing time compared to conventional methods.
- Cell viability of isolated ADSCs is 80–95%, significantly higher than traditional protocols.
- The method yields intact, morphologically normal adipocytes and uniform, viable ADSCs.
- Single-cell suspensions are suitable for primary culture, flow cytometry, cytotoxicity assays, and organoid construction.
Conclusions
- The combined enzymatic and mechanical wave-based protocol enables rapid, high-viability isolation of ADSCs.
- This approach reduces variability and preserves cell integrity, facilitating adipose tissue research and clinical applications.
- Future optimization may further improve isolation efficiency across different adipose depots.
Frequently Asked Questions
Executive Industry Relevance
Efficient and reproducible isolation of adipose-derived stem cells (ADSCs) is a critical inflection point for regenerative medicine, disease modeling, and cell therapy pipelines. The SoniConvert system's rapid, high-viability single-cell preparation addresses longstanding bottlenecks in cell sourcing, viability, and batch variability, directly impacting downstream assay reliability and translational research continuity. This capability enables more predictive early discovery and supports robust portfolio advancement decisions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rapid generation of high-integrity ADSCs for hypothesis-driven pathway interrogation.
- Reduces biological variability, supporting functional target validation and mechanistic de-risking.
- Improves predictive confidence for early-stage portfolio triage by standardizing cell input quality.
Screening & Assay Development
- Delivers single-cell suspensions compatible with primary culture, flow cytometry, and cytotoxicity assays.
- Supports assay reproducibility and quantitative output by minimizing cell damage and batch effects.
- Facilitates scalable preparation of validated biological systems for compound screening workflows.
Translational & Preclinical Research
- Provides high-viability cells suitable for organoid construction and disease-relevant modeling.
- Enables continuity from discovery through preclinical validation by preserving cell phenotype and function.
- Reduces risk of late-stage biological failure by ensuring consistent cell quality across studies.
Pipeline & Workflow Integration
This method integrates at the interface of tissue sourcing and early discovery, supporting workflows from target validation through assay development and translational research.
- Discovery Biology: Supports robust null hypothesis testing and pathway clarification by providing high-quality ADSCs.
- Screening: Ensures assay readiness and reproducibility with standardized, viable single-cell suspensions.
- Analytics: Enables quantitative viability and yield measurements for cross-condition comparison.
- Translational Research: Aligns with organoid and disease model construction for preclinical continuity.
- Enterprise Reuse: Establishes a reproducible, scalable cell isolation platform for diverse R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in cell-based studies.
- Operational Value: Standardizes isolation, improves reproducibility, and reduces processing time by over 90%.
- Strategic Value: Enables better go/no-go decisions and capital efficiency by minimizing batch variability and cell loss.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of regenerative and cell therapy assets.
Implementation Considerations
- Requires expertise in tissue handling, enzymatic digestion, and mechanical dissociation protocols.
- Needs access to the SoniConvert system, automated cell counters, and fluorescence microscopy infrastructure.
- Demands cross-team standardization of tissue preparation and viability assessment procedures.
- May require adaptation for depot-specific extracellular matrix differences in adipose tissue.
- Dependent on consistent reagent quality and instrument calibration for reproducible outputs.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:27
Porcine Adipose Tissue Harvesting and Preparation
1:36
Dissociation of Adipose Tissue for Cell Isolation
3:08
Isolation of Adipocyte and Stromal Vascular Fractions (SVF)
4:21
Analysis of SVF Cell Suspension and Adipocyte Samples
6:16
Results
7:23
Conclusion
