Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

The purpose of this procedure is to have an ex vivo model to evaluate bone remodeling and recreate the tumor-bone microenvironment using calvaria culture or co-culture with cancer cells. Most calvaria technique is an organ culture system using calvaria bones from neonatal mice to evaluate bone remodeling. You can test the therapeutic potential of diverse molecules, or even recreate the bone-tumor microenvironment when you use co-cultures with cancer cells.

All of that in a simple and easy assay in a short time and a low cost. The aim of this technique is to have a tool to evaluate bone regeneration in the cancer bone microenvironment, using an ex vivo model of mouse calvarial organ culture. First, select a mouse pup and place it under the hood.

We use calvaria from five-to seven-days-old mouse pups. Take the head with the syringe, cut the skin, and clear the scalp area enough for dissect the calvaria. Identify the sutures of the skull, sagittal, coronal, and lambdoids.

Make a make a straight cut along the lambdoid sutures, to about the level of the eye. Cut on each side, from the lambdoid sutures towards the coronal suture. At 45 angle, make another cut to connect the end of the cut made with the cut of the anterior fontanel.

It is extremely important to define the skull sutures, sagittal, frontal, coronal, lambdoid, to guarantee the correct tissue inclusion and histology analysis. With fine tips forceps, remove the calvaria, and put it on a Petri dish cover. With a scalpel, make a straight cut from the posterior fontanel along the sagittal suture through the coronal suture.

Two hemicalvarias are obtained. Pick each calvaria up with the forceps, and place them in a new Petri dish with PBS. For culture, place the hemicalvaria in a 24-well tissue culture plate, containing one milliliter of media, and incubate for 24 hours at 37 degrees.

We can also use primary calvarial cultures to reproduce the tumor-bone microenvironment for bone resorption. To do that, we co-culture calvarias with cancer cells, or with conditioned media from cancer cells. 24 hours later, remove the media and replace it with media containing the treatment that you would like to test.

If you wish to evaluate cancer cells and bone interactions, or recreate the tumor-bone microenvironment, pass the calvaria to a low cellular attachment plate, to avoid the attachment of the cancer cells to the plate. Trypsinize the cancer cells, count them, and place them carefully at the top of the hemicalvaria. You can also use conditional media from cancer cells, and use it to incubate the hemicalvaria.

Incubate for six to seven days at 37 degrees, with five percent CO2. On the third day, change the media and continue incubating. The number of cells using in the co-culture depends on the cancer cellular line.

You need, first, to optimize the number of cells that it is needed to induce a response in the one. After the culture, the calvaria pass through a process of fixation, decalcification, and paraffin-embedded. Further, the tissues are sectioned in a microtome, stained, and histomorphometric analysis is performed.

The inclusion of the calvaria can be tricky. To make sure that the tissue is protected during the inclusion, you can put the tissue between sponges or use a tissue paper or other absorbent paper before to put it in the cassette. For tissue processing, wrap the hemicalvaria in tissue paper.

Then, place it into an embedding cassette, and fix in neutral buffer in formalin, for 24 hours at four degrees. To decalcify the calvaria, place the cassette into a 10%EDTA for 48 hours at four degrees. Process the tissue cassettes with the hemicalvaria in an automatic tissue processor.

Follow a regular day rotation cycle, then, include in paraffin. For sectioning, cut four microtic sections with a microtome, mount the sections onto glass microscope slides, and stain with hematoxylin, eosin. Histology analysis was used to perform quantitative analysis of the bone surface and the bone remodeling areas.

The images of the calvaria on the seven days were analyzed using the imaging software. For quantitative assessment, first, define the area for the analysis. Look in the sections on the telopa where four X to identify the orientation and the sutures.

Define the coronal suture, and identify the long bone surface on one side, and then the short surface on the other. Under the 40X magnification, identify the coronal suture and move two or three optical fields away from the suture, along the long surface. Capture the image of this area for analyze.

You can use images software to analyze the structural integrity of the bone. Quantitative histomorphometric analysis for the thickness and the bone area of the hemicalvaria can be done. The correct calvaria analysis depends of a good embedding and proper orientation for consistent histological results.

Be sure to use at least three calvaria per experimental condition. Here, we can see the structure of the bone. In orange, the bone is observed.

We can also see the presence of the periosteum, ostocyte, endosteum, and another parts of the bone. In our case, we use insulin to increase bone remodeling. Compared with a control, we can see a significant increase in the bone area.

Here, we can see the effect of MDA-MB-231 cell line on model calvaria. We can see, compared with the control, that the presence of cancer cells induce osteolytic factors that stimulate bone destruction. We used calvaria model to measure bone regeneration and cancer cell-bone interactions by co-cultures with cancer cell and histology.

We also validate our data by quantitative real-time PCR. This ex vivo model have many advantage, like the three-dimensional organization and the cellular diversity of the bone are preserved, and the experimental conditions can be controlled. Besides, the model is simple, you can see results in a short period of time, and it is low-cost.

And it can be combined with other techniques, like quantitative real-time PCR, microscopy, and micro-CT.