Spin Column-Based Lysosome Isolation: A Mini Review

(Quanzhi Li Ph. D. Invent Biotechnologies Inc.)

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Abstract

Lysosomes are central regulators of catabolism, nutrient signaling, and immune and stress responses. Accurate characterization of lysosomal contents and function depends on efficient organelle enrichment. Traditional approaches, including differential centrifugation, density gradients, and immunocapture, are often time-consuming, require large amounts of input material, and may compromise organelle integrity. Recently, spin column–based lysosome isolation methods have emerged as a versatile alternative. This review synthesizes applications of spin column–based lysosome isolation across recent studies in diverse sample types—including immune cells, hepatocytes, neurons, adipocytes, and clinical tissues—and highlights the downstream assays enabled by this approach. We emphasize the practical and technical advantages over traditional methods, Collectively, the literature supports spin column–based lysosome isolation as a reproducible, small-input, and function-preserving workflow for lysosome-focused biological discovery and translational studies.

Introduction

Lysosomes play essential roles in cellular homeostasis, nutrient sensing, immunity, and cell death. Their functional diversity and dynamic remodeling necessitate methods that allow robust enrichment for biochemical, imaging, and omics analyses. Traditional approaches, such as differential centrifugation and density gradients (e.g., Percoll, OptiPrep), are widely used but can require large amounts of input material, are labor-intensive, and may co-fractionate contaminating organelles. Immuno-isolation techniques (e.g., LysoIP) improve specificity but require genetic modification or specialized antibodies and equipment.

Spin column–based lysosome isolation has emerged as a practical alternative, using a rapid filtration and selective precipitation to enrich lysosomes without ultracentrifugation or gradient media. The method is suitable for small samples and compatible with high-throughput processing. Here, we review unique applications of spin column–based lysosome isolation across various sample types and downstream assays, summarizing its comparative advantages.

Applications Across Sample Types and Downstream Assays

1. Immune Cells

Spin column–based lysosome isolation has been instrumental in studies of T cell biology and immune signaling. Edwards-Hicks et al. [10] used lysosome-enriched fractions from CD8⁺ effector T cells to perform phosphoinositide acyl-chain profiling by lipidomic (LC–MS), linking lipid composition with T cell activation. Qing et al. [11] targeted lysosomal HSP70 in T cell malignancies, using enriched fractions to measure acid sphingomyelinase activity and lipid metabolism changes. Tsujimoto et al. [9] demonstrated that lysosomal integrity measured in spin column fractions was essential for defining Regulator–HDAC6–mediated NLRP3 inflammasome activation in vivo.

2. Liver and Hepatocyte Models

Spin column–enriched lysosomes were pivotal in several studies of hepatocyte metabolism, hepatotoxicity, and hepatocellular carcinoma (HCC). Casey et al. [3] used lysosome fractions to contextualize lipid droplet proteome remodeling in ethanol-induced steatosis. Jing et al. [4] localized phosphoenolpyruvate carboxykinase (PCK) isoenzyme activity to lysosomes in liver cancer, while Tao et al. [8] used lysosomal membrane permeabilization (LMP) markers from fractions to define perphenazine-induced hepatotoxicity. Qian et al. [26] combined proteomics and FACS on lysosomal fractions to study CD147 internalization as a therapeutic vulnerability in HCC.

Spin column-based lysosome isolation also supported metabolomics of cancer metabolism under ketogenic diet conditions [22], antiviral mechanism studies of engineered antibodies in hepatitis B virus infection [20], and Lamp2a deficiency–induced bile acid dysregulation in autoimmune cholangitis [28].

3. Brain and Neuronal Systems

Lysosomal dysfunction is central to neurodegeneration and neuronal injury. Li et al. [12] and Yang et al. [23] leveraged lysosome-enriched fractions to study chaperone-mediated autophagy (CMA) in sepsis-associated encephalopathy and stroke, respectively. Secker et al. [14] used fractions to demonstrate lysosomal degradation of intracellular amyloid-β after polyphenol EGCG treatment, while Chen et al. [16] localized labile iron pools regulated by TFEB to lysosomes. Wang et al. [27] showed that lysosomal ABCA1-mediated cholesterol accumulation in APOE4 carriers contributes to cellular senescence in Alzheimer’s disease.

4. Adipose and Metabolic Systems

Spin column–isolated lysosomes were used to assess organelle integrity and signaling in adipocytes. Morishige et al. [17] demonstrated that Sphingosine kinase 1 maintains lysosomal integrity to enable triglyceride breakdown in brown adipocytes, while Ren et al. [13] used lysosome fractions to measure AMPK activity independent of PEN2 regulation.

5. Cancer Cell Lines and Targeted Drug Delivery

Kim et al. [2] used lysosome fractions to examine cathepsin A regulation in canine inflammatory mammary carcinoma, while Cao et al. [7] employed lysosome-enriched fractions for nascent glycoproteome analysis of LAMP2 regulation. Lin et al. [15] studied calcium flux and mitochondrial death after lysosomal delivery of doxorubicin using RBC-derived vesicles. Spin column lysosome fraction also supported studies of oxidative stress tolerance through cyst(e)ine storage [18], SNX10-mediated nutrient signaling [29], and ferroptosis resistance via protein S-glutathionylation [30].

6. Infection and Virology

Spin column fractions were applied in time-sensitive infection models, where workflow speed and reproducibility are essential. Wang et al. [19] demonstrated that betacoronavirus PHEV egresses through lysosomal intermediates, while Qian et al. [31] linked palmitoylation to CMA-mediated degradation of PEDV spike protein.

7. Clinical Samples

In human appendix tissue from Parkinson’s disease patients, Gordevicius et al. [5] used lysosomal fractions to quantify epigenetic repression of the autophagy–lysosome system. These studies illustrate the method’s suitability for scarce clinical material.

Advantages of Spin Column–Based Lysosome Isolation

Across studies, spin column–based lysosome isolation demonstrated consistent advantages over traditional methods:

  1. Speed and practicality: The workflow can be completed in <1 hour, avoiding ultracentrifugation and density gradients. This is advantageous for time-sensitive assays such as infection models [19] and immune activation studies [10].
  2. Small input requirements: The method accommodates limited starting material, including small clinical biopsies [5,12,23,27].
  3. Broad assay compatibility: Lysosomal fractions have been successfully used for mass spectrometry–based proteomics and lipidomics [3,7,10,26], enzyme activity assays [8,11,13], labile metal and ion measurements [15,16], flow cytometry [26], and imaging [14,19].
  4. Reproducibility and throughput: Standardized columns reduce operator variability and allow parallel processing of multiple samples, supporting comparative and high-throughput designs [8,10,26].
  5. Preservation of functional integrity: Fractions maintain lysosomal integrity for assays of LMP, enzyme activity, and CMA flux [8–9,12,23].
  6. Safety and accessibility: Spin columns avoid toxic gradient media and specialized equipment, facilitating adoption in standard laboratories [5,19].

Conclusions and Future Directions

Spin column–based lysosome isolation provides a robust, reproducible, and small-input workflow applicable across a broad spectrum of sample types and assays. The studies reviewed here collectively demonstrate its utility in elucidating lysosomal roles in metabolism, neurodegeneration, infection, immunity, and cancer biology. Future directions include automation for high-throughput multi-omics pipelines, deeper profiling of lysosomal subpopulations, and expanded application in clinical biomarker studies.

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