4.7 Article

Isolation of Viable Single Cells With High Yield and Purity Using a Small Amount of Human Kidney Tissue Biopsy

Journal

Publisher

FRONTIERS MEDIA SA
DOI: 10.3389/fcell.2022.822275

Keywords

human kidney; core needle biopsy; single-cell suspension; tissue dissociation; enzymatic digestion; cell purification

Funding

  1. National Natural Science Foundation of China [82170716]
  2. Key Research and Development project of Shanxi Province [201903D321086]
  3. Development and promotion of diagnostic kit for hereditary polycystic kidney disease based on second-generation sequencing technology of target gene [201903D321086]

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The study developed an efficient method for preparing single cell suspension from human kidney tissue puncture using digestive enzymes, achieving high purity and viability of cells.
Objective: Establishment of an efficient method of preparing human kidney single cell suspension, using a very small amount of tissue puncture. Methods: Samples of human kidney tissue puncture were cut into pieces, and then 80 mu L of the digestive enzyme were added to each punctured tissue to induce enzymatic digestion. The enzyme combination is composed of collagenases, DNase and hyaluronidase and the sample was incubated 20 min at 37 degrees C. The obtained cell suspension was filtered through a 70 mu m cell strainer, centrifuged at 300 g for 5 min and the supernatant was removed, then the pellet was resuspended in 3 ml of DMEM (Dulbecco's Modified Eagle's Medium). Cell suspension was sorted and purified by flow sorting to remove dead cells and obtain a cell suspension with higher viability rate. Results: We found that 1) diverse single cells of human kidney can be obtained by the digestive enzyme, as observed under the light microscope, with different sizes, normal cell morphology and good dispersion. 2) (2-3) x 10(6) single cells can be extracted from one fresh punctured kidney tissue of about 10 mg, with a cell viability rate of more than 80%. Conclusion: In this work we generated a comprehensive and high-resolution single-cell method, which is simple and efficient for preparing single cell suspension from a minimal amount of human kidney tissue. This method can facilitate the study of renal cell biology and the pathogenesis of kidney diseases.

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