How to get osteoclasts by different induction methods?

The skeletal system provides an important basis for supporting the body, coordinating body movement, controlling mineral homeostasis and hematopoietic production. Osteoclasts are specialized cells derived from the fusion of monocyte/macrophage hematopoietic lineage progenitors, and are essential for bone homeostasis and health maintenance. A large number of experiments have demonstrated that its cell formation and dysfunction are closely related to osteoporosis, fracture healing, osteoarthritis and primary/metastatic bone tumors. Osteoclasts, as "bone eaters", have been widely studied for their roles in bone homeostasis and disease.

Osteoclasts are terminally differentiated cells and cannot be passaged. Under microscope, osteoclasts are characterized by large cell bodies, multinucleated, pseudopodia and protrusions.

                                                                                           Osteoclast morphology

In experimental studies, osteoclasts are often obtained by inducing stem cells or monocytes in vitro to differentiate with the help of inducers. This method produces osteoclastoid cells. Osteoclastoid cells are primary or induced cells with osteoclastic properties, which have been widely used in the study of osteoclasts in recent years. The cells obtained by induction method have high purity and large quantity, which can satisfy various biochemical and meristem experiments. There are two common methods for osteoclast induction.

Primary cell induction

Currently, the most common cell line that can be induced to differentiate into osteoclasts is mouse RAW 264.7 cell line. Here, we will take this cell as an example to introduce its induction method.

AW 264.7 can be induced by LPS, RANKL, or M-CSF + RANKL.

1. LPS induction.

The concentration could be 10ng/mL, 30ng/mL, 50ng/mL, 100ng/mL. But 30ng/mL, 50ng/mL, 100ng/mL LPS induction will produce more multinucleated cells, and multinucleated cells through 30ng/mL LPS induction increase significantly in size. 

                                                               Osteoclasts induced by different LPS concentrations

2. RANKL induction

Inducing by α-MEM medium containing 100μg/L RANKL. To refresh the meidum every other day, and a large number of multinucleated cells will appear on the 4th to 5th day of induction culture. 

3. M-CSF + RANKL induction

Inducing by DMEM complete medium containing 100ng/mL RANKL and 100ng/mL M-CSF. To refresh the medium every two days, and osteoclasts will appear around the 4th day.

Osteoclast identification: TRAP staining

Tartrate-resistant acid phosphatase(TRAP) mainly reflects the activity and bone resorption capacity of osteoclasts, which is a good marker of osteoclasts and the most commonly used method for identification. Osteoclasts can be identified according to the following steps.

TRAP staining procedure:

Fix the cells with 2.5% glutaraldehyde for 10 min, fully wash with distilled water, treat with TRAP dye for 50 min, and then seal with glycerin after fully rinsed. Positive stained cells are red and located in the cytoplasm, while negative stained cells are yellow.  

                                                                                              Osteoclasts stained by TRAP

Notices 

a. The number and purity of mature osteoclasts induced by primary cells is limited, but bone resorption activity is strong. The number and purity of osteoclasts induced by RAW 264.7 cell line is high, but the absorption activity is weak.

b. During culture, if more cells showed pseudopodia under the microscope, the overall state of the cells may be poor, and these cells are not suitable for osteoclast induction.

c. The number of passage of RAW 264.7 cells is very important to the induction of osteoclasts. The lower the passage number, the higher the differentiation ability.

d. The induction cycle of osteoclast formation in primary cells is long, and it usually takes 9-10 days to reach the best state. The induction cycle of RAW 264.7 was shorter, and a large number of osteoclasts appeared on the 5th to 6th day.

e. In addition to TRAP staining, observing the number of nuclei, calcitonin receptor staining, and observing resorption lacunae of bone / teeth can also be used to further identify whether osteoclasts have bone resorption capacity.