Name: isolation of monocyte-macrophage lineage cells from rat bones by secondary adherence method
Uploaded: 2022-07-13T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method at JoVE.com

This work was supported by the Natural Science Foundation of Zhejiang Province (grant no. LY19H060001) and the Zhejiang Traditional Chinese Medicine Science and Technology Plan Project (no. 2022ZB093).

All experiments in this study were conducted in accordance with the animal experiment guidelines of the Zhejiang Chinese Medical University Laboratory Animal Research Center (Approval No: IACUC-20181029-11).
1. Cell extraction
<ol>
	<li>Put the Sprague-Dawley rats (SD rats, 1-10 days old, male or female) in the euthanasia cages filled with CO2 at a balanced rate of 30%-70% container volume/minute. After the rats lose consciousness (20-60 min), euthanize the rat by...

<ol>
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Osteoclasts are one of the most significant cell types involved in the occurrence and development of bone diseases, as well as one of the primary objects of bone disease research20. Monocyte/macrophages can differentiate into osteoclasts. Since mononuclear macrophages (RAW264.7 cells) are too expensive to buy and are easily activated during culture, it is difficult to perform in vitro differentiation experiments using this cell line. Although several methods have been developed for extrac...

It has been reported that monocyte-macrophage lineage cells existing in the bone marrow can differentiate into blood monocytes and tissue macrophages1,2. The above cells, which can differentiate into osteoclasts to balance bone growth and development, are commonly used as a cell model to induce osteoclasts in vivo3,4. Bone marrow is a special tissue containing several different types of cells, which include but are not only limited to bone marrow mesenchymal stem cells, bone marrow monocyte-macrophage cells (BMMs), hematopoietic stem cells, endothelial cells, and immune cells. In fact, several previous studies suggested that adherent cells rushed out of the bone marrow cavity of the long bone could differentiate into osteoblasts, osteoclasts, chondrocytes, or adipocytes5,6,7,8. Although, different isolation and culture methods have been used to produce different homogeneous cell populations, there are still great challenges in isolating and culturing BMMs from a variety of different cell types.
Several methods have been developed to extract bone marrow mononuclear macrophages (BMSCs). However, the majority of these methods are complex9,10,11. For example, density gradient centrifugation requires a specialized kit and the operation is time-consuming and cumbersome. This method is suitable for the isolation of BMMs from high-volume blood samples, but not from bone marrow samples9,12,13. In addition, extracting tissue samples using collagenase digestion is a complex and time-consuming procedure; this method is not recommended for the isolation of BMMs from bone marrow samples14,15. In addition, although flow separation can result in highly purified monocyte/macrophage populations, it requires very large sample sizes and high instrument and equipment requirements10,16. Additionally, the microbead enrichment method is extremely expensive and is not feasible in a general laboratory17.
Therefore, in the current study a convenient, fast, and cheap method was proposed for the isolation of mononuclear macrophages from the bone marrow. Bone marrow cells adhered for different time points were used to isolate BMMs using a secondary adherence method. BMMs extracted with the above method could induce the formation of osteoclasts in vitro, thus providing a simple and convenient cell model for the future study of osteoporosis in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm2 cell climbing slices</td>
			<td>NEST Biotechnology</td>
			<td>80102</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-cathepsin K</td>
			<td>Abcam</td>
			<td>ab19027</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-CD11 isotype control</td>
			<td>Abcam</td>
			<td>ab172730</td>
			<td>1 &#956;g/test,1.675 mg/Ml</td>
		</tr>
		<tr>
			<td>Anti-CD11b/c</td>
			<td>Absin</td>
			<td>abs124232</td>
			<td>&#160;1&#956;g/test, 1 mg/mL</td>
		</tr>
		<tr>
			<td>Anti-TRAP</td>
			<td>Abcam</td>
			<td>ab191406</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin</td>
			<td>Beyotime</td>
			<td>&#160;AF5003</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Cell climbing slices</td>
			<td>NEST Biotechnology</td>
			<td>80102</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td>for IF, 1:2,000</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td>for WB, 1:2,000</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Pepro tech</td>
			<td>400-28</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biosharp</td>
			<td>BL302A</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>Pepro tech</td>
			<td>400-30</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rat</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd</td>
			<td></td>
			<td>1-10 days old</td>
		</tr>
		<tr>
			<td>SDS-PAGE gel preparation kit</td>
			<td>Solarbio</td>
			<td>P1200</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP/ALP Staining Kit</td>
			<td>Wako</td>
			<td>294-67001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Biosharp</td>
			<td>BL512A</td>
			<td></td>
		</tr>
		<tr>
			<td>Wright-Giemsa solution</td>
			<td>Keygen Biotech</td>
			<td>KGA225-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of monocyte-macrophage lineage cells from rat bones by secondary adherence method

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

The secondary adherent cell population was stable and uniform. With the continuous cell proliferation, the majority of cells became larger, with irregular shape and grew into a radial adherent disk (Figure 2C,D). Flow cytometry showed that the percentage of cells expressing CD11b/c, a molecular marker on the surface of monocyte-macrophage lineage cells, was approximately 37.94% (Figure 2A,B). To ...

Watch this Scientific Journal Video about Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method at JoVE.com

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and ...

In this protocol, bone marrow monocyte-macrophage cells are extracted by secondary adherence method. Bone marrow monocyte-macrophage cells can successfully differentiate into osteoclasts, which provides a stable cell model for the study of osteoclasts in vitro. This method is suitable for small bone marrow samples and can extract bone marrow monocyte-macrophage cells from bone marrow simply and quickly without requiring high tech lab equipment.The increase of osteoclasts may be the potential pathogenesis of osteoporosis. Bone marrow monocyte-macrophage cells have certain research value as osteoclast precursor cells. To begin, immerse the euthanized rats in 75%alcohol for 10 minutes for disinfection.After carefully removing all the limbs of the rat with scissors and forceps, aspirate with PBS using a pipette and flush the blood adhering to the limbs. Then at two milliliters of the 10%FBS DMEM into a five milliliter tube. After transferring the limb bones into the five milliliter tube, use scissors to cut the limb bones in the tube into small pieces and mix the homogenate to resuspend the bone marrow cells into the culture medium.Stand for five minutes until the tissue fragments settled to the bottom of the tube. Next, add 10 milliliters of 10%FBS DMEM into a 100 millimeter culture dish, and then transfer the supernatant into the culture dish. Incubate at 37 degrees Celsius in 5%carbon dioxide for approximately 24 hours.After this incubation time, the majority of mesenchymal stem cells will adhere to the culture dish wall and grow slowly, while most bone marrow monocyte-macrophage cells will still be suspended in the culture medium. Transfer the cell suspension in the 100 millimeter culture dish to a new 25 centimeter square flask and continue to cultivate the cells at 37 degree Celsius in 5%carbon dioxide for an additional 24 hours. After removing the old medium carefully, replace with fresh medium after bone marrow monocyte-macrophage cells have adhered to the flask wall.Flow cytometry showed that the percentage of cells expressing CD11b and c, a molecular marker on the surface of monocyte-macrophage lineage cells was approximately 37.94%With the continuous cell proliferation, the majority of cells became larger within irregular shape and grew into a radial adherent disc. The TRAP stain results showed that compared with the control group, the number of intracellular purple red granules was significantly increased in cells induced with receptor activator of nuclear factor kappa B ligand and macrophage colony stimulating factor. Western blot analysis demonstrated that the expression levels of the osteoclast specific proteins TRAP and cathepsin K were significantly increased in treated cells compared to the control group.The most important thing in this procedure is the time to collect secondary adherent cells in 24 to 48 hours of primary culture, bone marrow monocyte-macrophage cells begin to adhere completely. Bone marrow monocyte-macrophage cells isolated by this method can be induced into osteoclasts in vitro, providing a stable cell model for the study of osteoporosis.

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