Why Human Chondrocytes Morphologically Change After Passaging

Cartilage tissue comprises chondrocytes, matrix, and fibers, with human chondrocytes being isolated from cartilage tissue. Chondrocytes reside in cartilage lacunae.

Under electron microscopy, chondrocytes exhibit protrusions and folds, with abundant rough endoplasmic reticulum, a developed Golgi apparatus, and sparse mitochondria in the cytoplasm. In tissue sections, chondrocytes shrink into irregular shapes, creating sizable cavities between the cartilage capsule and the cells.

Culturing chondrocytes in vitro?is essential for studying their physiological functions, drug responses, and pathological changes under various pathogenic conditions.

Basic Information:

• Culture Medium: Contains FBS, growth supplements, and antibiotics.
• Media Change Frequency: Every 2–3 days.
• Growth Characteristics: Adherent growth.
• Cell Morphology: Spindle-shaped, polygonal.
• Passage Properties: Can be passaged up to 5 generations, with optimal conditions within the first three.
• Culture Conditions: Air: 95%; CO?: 5%.

Chondrocytes often undergo morphological changes during passaging, a common issue for many researchers. However, these changes are normal. Below is a detailed explanation of the morphological transformation process:

Morphology of First-Passage Cells

In the first passage, human chondrocytes exhibit spindle-shaped or polygonal morphology (Figure 1).

Morphology of Second-Passage Cells

After one passage, cells start to change shape, extending filamentous pseudopodia and resembling fibroblasts (Figure 2).

From the first to the second passage, the cells already display morphological changes. Will these changes persist with further passaging?

The answer is yes. By the third passage, fibroblast-like characteristics become more pronounced, with cells appearing long and spindle-shaped, and their growth rate begins to decline. These changes are more apparent by the fourth passage, and by the sixth passage, almost all cells morph into long spindle-shaped fibroblast-like cells. Therefore, second or third-passage chondrocytes are recommended for experiments.

Causes of Morphological Changes in Passaged Human Chondrocytes

In vitro, chondrocytes exhibit adherent growth. Primary cells are generally oval or polygonal. With increased passage numbers, the cells gradually become elongated and spindle-shaped, transitioning into fibroblast-like cells. Simultaneously, their function also changes: secretion of type II collagen, a chondrocyte-specific marker protein, decreases, while synthesis and secretion of type I and III collagen increase.

Type II collagen synthesis is a characteristic feature of differentiated chondrocytes, comprising 85%–90% of the collagen in hyaline cartilage. It provides cartilage with its unique tensile strength and rigidity. Other cartilage molecules interact with collagen to form a reticular structure, conferring elasticity to the cartilage and maintaining its shape while bearing external loads. As the number of passages increases, substances that maintain cell morphology gradually diminish, leading to morphological changes.

This phenomenon, known as dedifferentiation, is common in monolayer passaged cells.

Culture Tips:

1. Chondrocytes cultured at low densities are prone to dedifferentiation. The optimal seeding density is generally 2–10×10? cells/cm2.

2. Chondrocytes exhibit relatively slow metabolism, and increasing media change frequency does not enhance proliferation. Instead, it may disrupt the cell-secreted communal chemical environment.

Reference

Claus S , Aubert-Foucher E , Demoor M , et al. Chronic exposure of bone morphogenetic protein-2 favors chondrogenic expression in human articular chondrocytes amplified in monolayer cultures[J]. Journal of Cellular Biochemistry, 2010, 111(6).