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Cell Dissociation Techniques in Salamanders.

Gabriela Johnson¹, Nadjib Dastagir¹, Zachary Beal¹

¹Mount Desert Island Biological Laboratory. (MDIBL) Kathryn W. Davis Center for Regenerative Biology and Aging, Salisbury Cove, ME, USA.

Methods in Molecular Biology (Clifton, N.J.)

|October 22, 2022

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Choosing the right cell dissociation method is crucial for accurate tissue analysis. Different protocols impact cell recovery and surface marker expression, affecting downstream applications like flow cytometry and single-cell RNA sequencing.

Area of Science:

Cell Biology
Biotechnology
Regenerative Medicine

Background:

Cell dissociation is a critical step in analyzing tissue phenotypes.
The chosen method for cell harvesting significantly influences cell type recovery and introduces potential biases in cell surface protein phenotypes and gene expression.

Purpose of the Study:

To evaluate cell surface phenotypic changes and cell yields under four different dissociation conditions (enzymatic/non-enzymatic).
To assess the impact of dissociation methods on cell viability and diversity in axolotl spleen and regenerating limb tissues.
To provide a template for optimizing cell dissociation protocols for specific tissues and downstream applications.

Main Methods:

Compared four dissociation protocols: enzymatic (Liberase-DH, Liberase-TL, TrypLE) and non-enzymatic.

Keywords:

Axolotl Cell dissociation Phenotyping RNAseq Regeneration Salamander Single cells

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Evaluated cell liberation, viability, and cell diversity using live-cell flow cytometry.

Assessed cell surface marker expression and cell yields from axolotl spleen and regenerating limb tissues.

Main Results:

TrypLE and non-enzymatic methods yielded the highest surface marker expression but poor recovery of non-immune cells from the blastema.
Liberase-DH and Liberase-TL provided acceptable dissociation of diverse blastema cell types, with Liberase-TL yielding the most cells.
Enzymatic dissociation in the spleen resulted in lower yields, reduced viability, and significant loss of immune cell surface markers compared to non-enzymatic and TrypLE methods.

Conclusions:

Dissociation methodology critically affects cell recovery, viability, and phenotype, influencing downstream analyses.
Optimizing cell dissociation protocols is essential for balancing cell yield with the preservation of cellular integrity and marker expression.
The findings offer a framework for selecting appropriate dissociation methods for diverse tissues in applications like single-cell RNA sequencing and flow cytometry.