Isolation of Primary Cells from Animal Tissues Using Trypsin: Key Operational Points and Common Pitfalls

From Animal Tissue to Cell Suspension

In animal experiments, it is sometimes necessary to obtain cells directly from the liver, lungs, kidneys, and other organs of rats, mice, or rabbits. Cells isolated directly from living tissue in this way are called "primary cells." In tissue, cells are wrapped by connective tissue, collagen, and other substances, as if stuck together by "glue." Trypsin can break down some of the linker proteins, digesting tissue blocks into smaller fragments and further yielding dispersed cells.

Tissue Collection Must Be Fresh

After the animal is sacrificed, the target tissue should be removed as soon as possible and placed in pre-chilled buffer or culture medium, removing blood contamination and unwanted capsules. Fresh tissue responds more uniformly to digestive enzymes. Generally, the tissue is cut into small pieces of about 1–2 mm to increase the surface area exposed to trypsin. This step is not complicated; the key is to maintain low temperature to reduce cell damage.

Digestion Conditions Need to Be Optimized

The tightness of different tissues varies greatly. Tissues rich in fibers, such as muscle and skin, require longer digestion times, while softer tissues such as liver and spleen require shorter digestion times. Laboratories commonly use trypsin at a concentration of 0.1%–0.25%, incubating at 37°C for 10–30 minutes, during which gentle stirring or shaking may be applied. If the tissue blocks become noticeably looser and the liquid becomes turbid, it indicates that cells have detached.

Combine with Gentle Mechanical Dispersion

Trypsin alone sometimes cannot completely disperse the tissue. After digestion, a pipette can be used to gently triturate, or a sterile sieve can be used to gently grind, helping cells release from the tissue blocks. The mechanical force must be gentle, because cells freshly isolated from tissue are relatively fragile, and excessive force will significantly increase the proportion of dead cells.

Avoid Over-Digestion

Over-digestion will reduce the viability of the obtained cells. During digestion, a small amount of liquid can be taken every few minutes and observed under a microscope. When an increased number of single cells is seen and the edges of tissue blocks become frayed, digestion should be immediately terminated with serum-containing culture medium. Afterwards, undigested tissue blocks are filtered out using a sieve, and cells are collected by centrifugation. The whole process is like "disassembling" a piece of tissue; one must disassemble while observing, and cannot leave it unattended.

Common Misconceptions

Soaking directly in high-concentration trypsin overnight: This easily leads to cell death and is not recommended as a routine method.

Not changing the liquid throughout the process: Digestion products and dead cells will accumulate, affecting the state of viable cells.

Ignoring temperature control: Too high or too low a temperature will alter enzyme activity.

Summary

Trypsin is an important tool for obtaining primary cells from animal tissue. By controlling tissue block size, digestion time, and timely termination, ordinary experimenters can gradually master this technique, providing cells that are closer to the in vivo state for animal experiments.