The growth process of cultured cells: incubation period → exponential growth period → stagnation period.
I. Incubation period:
After the cells are inoculated, they go through a suspension period, during which they are suspended in the culture medium. At this time, the cytoplasm contracts and the cell body is spherical. Then the cells adhere to the surface of the carrier, which is called adhesion, and the suspension period ends. The adhesion speed of cells is closely related to the cell type, the composition of the medium and the physical and chemical properties of the carrier. In general, the adherent speed of primary cultured cells is slow, which can reach more than 10-24 hours, while the adherent speed of subcultured cells is fast, which generally takes 10-30 minutes. After cells adhere to the wall, they need to go through an incubation period before they can enter the growth and proliferation period. The incubation period of primary cultured cells is about 24-96 hours or longer, while the incubation period of continuous cell lines and tumor cells is shorter, only 6-24 hours.
Second, the exponential growth stage.
This is the most vigorous stage of cell proliferation, with more mitotic cells. The number of mitotic stages in exponential proliferation period can be used as an important index to judge whether the cell growth is vigorous or not. Usually expressed by mitotic index (MI), that is, the number of mitotic phases per 1000 cells in a cell population. The average cell division index is between 0. 1%-0.5%, which is the same as that of the original.
The mitotic index of progeny cells is low, while that of continuous cells and tumor cells can be as high as 3%-5%.
Exponential proliferation of cells is the best period of cell vitality, the best period of conducting various experiments, and the best time to freeze cells. When the number of inoculated cells is appropriate, the exponential proliferation period lasts for 3-5 days. With the increase of the number of cells and the decrease of the growth space, the cells finally contact each other and merge into blocks. Normal cells can inhibit cell movement after contact with each other, which is called contact inhibition. However, malignant tumor cells can continue to move and proliferate without contact inhibition, which leads to the expansion of cells into three-dimensional space and the accumulation of cells. After the cells contact and merge into blocks, although the contact inhibition occurs, as long as the nutrition is sufficient, the cells can still proliferate and divide, so the number of cells is still increasing. However, when the cell density further increases, the nutrients in the culture solution decrease and the metabolites increase, cell division stops due to the depletion of nutrients and the action of metabolites, which is called density inhibition.
3. Stagnation period
After the number of cells reaches the saturation density, if they are not passaged in time, the cells will stop proliferating and enter a stop period. At this time, the number of cells is flat, so it is also called platform period. Stagnant cells do not proliferate, but they still have metabolic activities. If cells are not separated and passaged, they will be poisoned by factors such as nutrient exhaustion, metabolite accumulation and pH drop, resulting in morphological changes, adherent cells falling off, and even death in severe cases. Therefore, it is necessary to pass on in time.
Basic morphological characteristics of cultured cells
The shape of cultured cells varies with the shape of the attached support, and the most common cells are attached to the planar support. Under the ordinary optical microscope, the living cells are homogeneous and transparent, and the structure is not obvious. There are often 1-2 nucleoli in growing cells.
When the cell function is not good, the cell contour will be enhanced and the contrast will increase. If granules, droplets and vacuoles sometimes appear in the cytoplasm, it indicates that the cell metabolism is poor.
According to the growth characteristics of cells cultured in vitro whether they can be attached to scaffolds, they can be divided into attached growth and suspended growth. Adherent cells can adhere to the surface of the support and grow in culture. For example, amniotic fluid cells are adherent cells, which often show the growth of fibroblasts and epithelial cells. Suspended cells grow in suspension in culture medium.
(1) fibroblasts
When the cell morphology is similar to that of fibroblasts, the cultured cells can be called fibroblasts. These cells are named because their morphology is similar to that of fibroblasts in vivo. Cells grow in spindle or irregular triangle on the surface of the support. There is an oval nucleus in the center of the cell, and the cytoplasm protrudes 2-3 cm, with different lengths. Except true fibroblasts, all tissue cells derived from mesodermal matrix often grow in this shape.
(2) Epithelial cells
The growth of this kind of cells on the support of culture container has the characteristics of flat and irregular polygon. There is a round nucleus in the center of the cells, and the cells are closely connected and grow like a single membrane. Cells derived from endoderm and ectoderm, such as skin, epidermal derivatives and digestive tract epithelium, all grow in the form of epidermis during culture.
(3) Wandering cells
This type of cell grows scattered on a support and is usually not connected into a block. The cytoplasm often extends out of pseudopodia or protuberance, showing active wandering or deformation movement, which is fast and irregular. These cells are not very stable, and sometimes it is difficult to distinguish them from other types of cells. Under certain conditions, due to the increase of cell density, it can be similar to polygons or fibrillated into fine packages. Common in the early stage of amniotic fluid cell culture.