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Primary cells are cells that are directly isolated from tissues. They not only maintain the morphology of normal cells, but also retain the key biological markers and functions in the body. Compared with cell lines, the experimental data provided by primary cells are closer to the real physiological environment, so they are of great value in biomedical research.

Because their biological properties are basically unchanged, primary cells can more accurately simulate the growth characteristics of the body and have been widely used in molecular biology, cell biology and basic biomedical research, such as proteomics, genomics, cell research and genetic research. At the same time, they are also ideal cell models in the field of biopharmaceuticals and are widely used in disease mechanism research, drug screening, drug metabolism, toxicology evaluation, cancer drug research and treatment development.

Cell extraction and in vitro culture refers to the process of isolating cells from tissues or organs and culturing them under appropriate conditions. The specific steps include: obtaining the target tissue under sterile conditions, using digestive enzymes such as trypsin or collagenase to dissociate the tissue into a single cell suspension, then separating the cells by centrifugation, filtration, etc., and finally culturing them in a suitable culture environment to maintain their growth and function.

Below, we will share the specific steps for primary cell isolation using a low-speed refrigerated centrifuge:

Experimental steps (digestion culture method)

Tissue preparation and washing

Take about 1 cm³ of target tissue under sterile conditions, place it in a plate or beaker, and repeatedly rinse it with an appropriate amount of Hanks solution to remove blood residues and connective tissue.

Tissue mincing and pretreatment

Use sharp scissors to mince the tissue into small pieces of approximately 0.5 mm × 1 mm, rinse again with Hanks solution, and discard the washing solution after the tissue pieces are settled.

Enzyme digestion

Transfer the tissue pieces to a conical beaker pre-installed with a sterile magnetic stirrer and add 5-30 mL of trypsin or collagenase.

Tightly cover with a rubber stopper and seal with tin foil, and place on a magnetic stirrer in a 37°C incubator.

Start the stirrer and stir evenly. Use a microscope to observe the cell dispersion during the digestion process.

When most cells are dissociated into a single state (about 10-20 minutes), immediately add an appropriate amount of Hanks solution to terminate the digestion.

Cell filtration

First, filter with a 100 μm stainless steel mesh to remove undigested tissue or large cell clusters.

Then filter with a 20 μm stainless steel mesh to obtain a relatively pure single cell suspension.

Low-speed centrifugation and cell washing

The filtrate was transferred to a centrifuge tube and centrifuged at 500 × g (about 5 minutes).

The supernatant was discarded and the cells were washed twice with Hanks solution to remove residual digestive enzymes.

The cells were resuspended in serum-containing medium and gently shaken to prepare a cell suspension.

Cell counting and inoculation

Use a hemocytometer to count and determine the concentration of the cell suspension (usually 1×10⁵~3×10⁵ cells/mL).

Evenly inoculate the cells into a culture dish and culture in a 37°C, 5% CO₂ incubator.

This method can effectively obtain highly viable primary cells and provide a reliable cell model for subsequent experiments. The use of a low-speed refrigerated centrifuge can help improve the separation efficiency of primary cells while ensuring cell viability, providing high-quality cell samples for subsequent experiments.

Notes

Tissue rinsing

Before digestion, tissue blocks need to be rinsed 2-3 times to remove the inhibitory effects of calcium, magnesium ions and serum on trypsin and EDTA.

Hanks solution or other appropriate buffer can be used to gently shake or stir the tissue blocks in the rinsing solution, and the rinsing solution can be discarded after the interfering substances are fully removed to ensure the effect of enzyme digestion.

The role of digestive enzymes

Trypsin is a proteolytic enzyme secreted by the pancreas. It mainly acts on the peptide bonds connecting lysine and arginine, breaking down proteins into smaller polypeptides and amino acids, which helps cells disperse.

Collagenase is derived from bacteria and has a strong ability to hydrolyze collagen. It can effectively digest fibrous tissue, epithelial tissue, and even some cancer tissues.

Improve cell survival rate

Properly increasing the seeding density of primary culture cells can promote the interaction between cells and make them closer to the in vivo growth environment, thereby improving cell survival rate and promoting adhesion and proliferation.

The key role of centrifuge in primary cell separation experiments

In the process of primary cell separation, low-speed refrigerated centrifuge plays a vital role, mainly used for cell enrichment, removal of digestive enzymes and debris, and improving cell survival rate. Its key applications in experiments include:

Cell enrichment and collection

After digestion, the tissue will be dissociated into a single cell suspension, but it may be mixed with incompletely digested tissue fragments, digestive enzyme residues and cell fragments. By properly setting the low-speed refrigerated centrifuge, intact cells can be effectively precipitated, enzymes and debris in the supernatant can be removed, and a relatively pure cell suspension can be obtained.

Wash cells to remove residual digestive enzymes

Digestive enzymes such as trypsin and collagenase may affect the normal function of cells or even be toxic to cells. Therefore, after the first centrifugation, the cells need to be resuspended in serum-free medium or Hanks solution and washed twice, each time centrifuged at a low speed and the supernatant discarded to ensure that most of the residual enzymes are removed and the cell survival rate is improved.

Cell concentration and optimization of culture conditions

Before inoculation, cells can be further concentrated by low-speed centrifugation to increase the inoculation density. Appropriate cell density not only facilitates cell-to-cell interaction, but also promotes adhesion and proliferation, thereby increasing the survival rate of primary cells and the success rate of culture.

Low-speed refrigerated centrifuges not only play a key role in the separation of primary cells, but are also widely used in many fields such as cell culture, clinical medicine, biopharmaceuticals, molecular biology and blood processing. Its low speed and efficient temperature control capabilities make it an ideal choice for temperature-sensitive sample processing. When using a centrifuge, the centrifugation parameters and operating procedures should be reasonably optimized to improve the success rate of the experiment and ensure the integrity of the sample and the stability of the experimental results.