Methods for Collecting Primary Cell Samples

Most tissues from humans and animals can be cultured in vitro, but the ease of culturing depends on several factors, including tissue type, level of differentiation, donor age, and the methods used for primary culture. Collecting primary samples is the first and most crucial step in tissue cell culture, as it directly impacts the success of subsequent experiments. In this session, we will outline the key requirements and methods for collecting different types of tissue samples.

Basic Requirements for Sample Collection

Tissue should ideally be processed into cells within 4–6 hours after collection. If immediate processing is not possible, the tissue can be soaked in culture medium and stored in an ice bath or at 4°C in a refrigerator. If the tissue is large, it should be cut into smaller pieces (less than 1 cm³) before low-temperature storage, but the storage time should not exceed 24 hours.

Strict aseptic techniques must be followed during sample collection. Sterile containers or pre-sterilized vials with a small amount of culture medium (containing penicillin and streptomycin) should be used to transport the samples.

When collecting and processing primary cultures, use sharp instruments such as scalpels to finely cut the tissue, minimizing mechanical damage to the cells.

Blood, fat, nerve tissue, connective tissue, and necrotic tissue should be carefully removed during sample collection. To prevent tissue desiccation during separation, the procedure should be performed in a culture dish with a small amount of culture medium.

Primary cultures, particularly those of normal cells, should use complete growth media.

In general, embryonic tissue is easier to culture than tissue from mature individuals, and less-differentiated tissues grow more readily than highly differentiated ones.

To facilitate later identification of the primary tissue source and to observe the differences between the cultured cells and the original tissue, histological and electron microscopy samples should be collected alongside the primary tissue samples.

Basic Equipment and Supplies for Sample Collection

1. Ophthalmic curved scissors, curved forceps, and scalpels (sterilized before use)

2. Small vials containing either serum-free medium or Hank’s solution

3. Small beakers (10 mL, 50 mL)

4. Culture dishes

Methods for Collecting Samples from Various Tissues

Collection of Mouse Embryo Tissue

Because mouse fur commonly harbors microorganisms and is difficult to disinfect, it is crucial to maintain strict aseptic conditions during sample collection. The following methods are commonly used:

1. Use the cervical dislocation method to euthanize the animal; then submerge the entire body in a beaker containing 75% ethanol for 3–5 minutes. Be mindful not to exceed this time, as ethanol may enter the body through the mouth and other orifices, affecting tissue viability.

2. Remove the mouse and place it on a sterilized dissection board, using sterilized pins or tacks to secure it.

3. Use ophthalmic scissors and hemostats to open the skin and collect the samples.

4. Alternatively, after ethanol disinfection, a circular incision can be made around the middle of the animal’s body, and the skin can be peeled back toward the head and tail using hemostats. Once the body is exposed, the sample can be collected and placed in a sterile culture dish or on a clean glass plate for primary culture. All post-disinfection procedures should be performed in a biosafety cabinet or sterile environment.

Collection of Chicken Embryo Tissue

When working with chicken embryos, it is common practice to incubate them yourself. The main steps are as follows: Select fresh, fertilized eggs, clean the surface, and incubate at 37°C in a standard incubator. Place a dish of water inside the incubator to maintain humidity. Typically, 9–12-day-old chicken embryos are used, and the eggs should be turned once a day during this period. Under sterile conditions, place the egg with the air sac (large end) facing up in a small beaker, and disinfect with iodine and ethanol. Use scissors to make a circular cut around the air sac end, open the shell membrane, and expose the embryo. Gently lift the embryo out with a blunt glass rod and place it in a sterile culture dish for sample collection.

Collection of Skin and Mucosal Samples

Skin and mucosal tissues are usually obtained from parts excised during surgery. If needed, they can also be collected independently. The method is similar to the operation used in dermatological surgery to excise tissue layers, but the area is generally only 2–3 mm², leaving no visible scar. Note: Do not disinfect the sample area with iodine. If necessary, wash with a high-concentration antibiotic solution.

Collection of Visceral and Solid Tumor Samples

When collecting visceral and solid tumor samples, it is essential to know and be familiar with the specific tissue type and location you need. Unnecessary parts, such as blood vessels, nerves, and connective tissue between organs, should be trimmed away. For tumor samples, collect the areas with the highest concentration of tumor cells, avoiding necrotic and liquefied regions.

Collection of Blood Cells

Venous blood is usually drawn, although small quantities can be obtained from the fingertip or earlobe. To prevent clotting, heparin is often used as an anticoagulant, with the minimal amount necessary to achieve an anticoagulant effect. Excessive heparin can lead to hemolysis. A common concentration for heparin is 20 U/mL, and the syringe should be pre-moistened with a higher concentration of heparin (500 U/mL) before drawing blood. Strict aseptic conditions should be maintained during blood collection.

Collection of Cells from Bone Marrow, Amniotic Fluid, Pleural Effusion, and Ascites

These samples generally require no additional processing after collection. After centrifugation, they should be cultured immediately, as low-temperature storage is not recommended.