Primary cell isolation
Minced tissue is digested with collagenase (often with dispase or trypsin) and DNase at 37 °C until it falls apart, then filtered and washed to give a single-cell suspension of the tissue's cell types.
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Enzymatic dissociation
Minced tissue is digested with collagenase (often with dispase or trypsin) and DNase at 37 °C until it falls apart, then filtered and washed to give a single-cell suspension of the tissue's cell types.
How it differs from the standard method
- Mince to about 1 mm; the enzyme works in from the surface of each piece.
- Use a buffer with calcium and magnesium (HBSS) for collagenase, and stop with serum-containing medium or EDTA.
- Pipette the digest every 10–15 min and stop when the pieces have broken up, not at a fixed time.
- Gentler, shorter digests keep surface markers for flow; longer digests release more of the matrix-bound cells.
1Taking cells straight from the body
A primary culture starts from cells that were part of an animal or a person hours earlier, rather than from an immortal line that has adapted to plastic over decades. That is the point of it: primary cells keep the receptors, metabolism and responses of their tissue.
Dissociation
Collagenase, dispase or trypsin digest the matrix of minced tissue; DNase stops dead cells' DNA gluing the rest together.
Density gradient
Mononuclear blood cells float on Ficoll; red cells and granulocytes sink through it.
Explant
Small pieces stuck to the dish release migrating cells over one to three weeks.
2What decides yield, viability and purity
In a digest, the enzyme works from the surface of each piece inwards, so mincing finely does more than anything else to speed it up, and cells released early keep being digested until you stop. Too short a digest leaves tissue on the strainer; too long or too strong kills the cells you already have and strips proteins off their surface.
3How it goes wrong
- Coarse mincing or a calcium-free buffer: most of the tissue is still in lumps after the digest.
- Strong enzyme for a long time: high yield on paper, low viability and missing surface markers.
- No DNase: free DNA ties cells into strings and clumps that are lost on the strainer or clog a cytometer.
- An unperfused organ: the suspension is mostly blood.
- A cold gradient, brake on, or blood more than a day old: granulocytes and red cells in the PBMC fraction.
- No slow wash after Ficoll: platelets stay with the PBMCs and clump them.
- Explants flooded at once or moved every day: the pieces float and nothing grows out.
- Skin or gut tissue without antibiotic washes: the dish is contaminated before any cells appear.
Key terms
Practise these as flashcards →Cells grown directly from tissue, before the first passage; they have a finite lifespan and keep many tissue-specific properties.
A calcium-dependent metalloprotease that cleaves collagen in the extracellular matrix; sold in types with different side activities.
An enzyme that degrades free DNA released by dead cells, preventing the sticky strands from clumping the suspension.
Peripheral blood mononuclear cells: lymphocytes and monocytes, the cells with a single round nucleus that collect at a Ficoll interface.
A density medium of sucrose polymer and sodium diatrizoate at 1.077 g/mL, used to separate mononuclear cells from blood.
The pale layer of white cells between plasma and red cells in spun blood, often used as a richer starting material for PBMC isolation.
A small piece of tissue placed in culture so that cells grow out of it onto the dish.
Flushing an organ's blood vessels with buffer before removal, to clear blood cells from the tissue.
When to use it
- When the question depends on how a real tissue's cells behave, and a cell line would not have the right receptors, metabolism or genotype.
- When cells must come from a specific patient, donor or mouse strain, for example immune cells for a functional assay or fibroblasts for reprogramming.
- When a cell type has no good immortal equivalent, or when results from a line need confirming in primary cells.
Compared with alternatives
Once isolated, primary cells are passaged like any culture, but they senesce after a limited number of doublings, so passage numbers stay low.
Every isolation ends with a count and a viability; it is how you know whether the isolation worked and how many cells to plate.
When you need one defined population out of the mixture an isolation gives, sorting or bead enrichment follows the isolation.
How the tissue or blood is collected and transported sets the ceiling on what any isolation can recover.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Minced tissue is digested with collagenase (often with dispase or trypsin) and DNase at 37 °C until it falls apart, then filtered and washed to give a single-cell suspension of the tissue's cell types.
- Mince to about 1 mm; the enzyme works in from the surface of each piece.
- Use a buffer with calcium and magnesium (HBSS) for collagenase, and stop with serum-containing medium or EDTA.
- Pipette the digest every 10–15 min and stop when the pieces have broken up, not at a fixed time.
- Gentler, shorter digests keep surface markers for flow; longer digests release more of the matrix-bound cells.