Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage

Abstract

Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague-Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Then, to evaluate LCM-RNA integrity, 3'/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3'/m ratio means of GAPDH and β-Actin ranged between 1.11-1.56 and 1.41-2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated.

Figure 1

Tissue procurement from the experimental model. (a) Five-week-old male SD rats with the TMJ and femoral joint encircled. (b) Mandibular ramus dissected with the mandibular condyle encircled. (c) MCC with minimal condylar bone was harvested. (d) Femoral bone dissected with the femoral condyle encircled. (e) FCC without the underlying bone was harvested.

Figure 2

Schematic workflow for the experiment of isolating homogenous cell populations from FCC and MCC zones using the LCM technique.

Figure 3

PixCell II laser capture microdissection instrument: (a) infrared laser and inverted light microscope, (b) laser control tower, (c) PC monitor with live video display, and (d) video camera.

Figure 4

Map images of stained MCC cryosections with 100 μm grid overlay. (a) Under 4x objective. (b) Under 10x objective using HistoGene Staining Solution.

Figure 5

Empirical setting of LCM parameters. (a) A properly melted polymer spot has a dark outer ring and a clear center. (b) Underexposed polymer spot has a fuzzy appearance and lacks the dark ring. (c) Laser overexposure may lead to polymer burning.

Figure 6

Adjustment of LCM parameters allows capturing of different sizes/areas of tissue. (a) MCC tissue section is prepared for LCM. (b) The section after LCM, where clusters of cells removed. LCM power and duration were adjusted to avoid burning (arrow) of the cap film. (c) Proper wetting permits the film to contact the selected area of the tissue section. (d) The section after LCM demonstrating capturing of a strip (continuous line) of tissue and (e) capturing of a larger zone. (f) After LCM completion, the LCM cap is examined to confirm successful microdissection of the cells of interest (scale bar = 100 μm).

Figure 7

Inspection of the cap surface upon LCM completion to verify the successfully performed procedure. Microdissected tissue can be seen with the naked eye (a) and/or under the LCM microscope (b and c).

Figure 8

Comparison of images taken for stained FFPE (a and c) and frozen (b and d) tissue sections demonstrated equally clear morphological details that permit identification of the different cell populations of MCC (scale bar = 100 μm).

Figure 9

Images of stained (a, b, and c) and unstained (d, e, and f) frozen sections of MCC tissue taken directly on the LCM instrument without coverslip; both types of sections allowed clear distinction between MCC zones (scale bar = 100 μm).

Figure 10

Zonal isolation of homogenous cell populations from the MCC tissue. (a) Index-matched image is used to guide the LCM process. (b) Unstained, dehydrated, frozen MCC tissue section before LCM demonstrating the four zones (FZ, MZ, PZ, and HZ). (c–f) MCC sections after LCM where cells were individually isolated from their respective zones.

Figure 11

3′/m ratio determined by RT-qPCR as a measure of the quality of LCM-RNA samples for GAPDH.

Figure 12

3′/m ratio determined by RT-qPCR as a measure of the quality of LCM-RNA samples for β-Actin.