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

Aisha M Basudan,Yanqi Yang

doi:10.1155/2019/5427326

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.

Highlights

Articular surfaces of the temporomandibular joint are covered by fibrocartilage, and the one covering the condyle is known as mandibular condylar cartilage (MCC)
We selected the age of 5 weeks because MCC articulation function is already present in a more mature state and because the maximum growth spurt for rats occurs at day 31.5. e use of these animals was approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong (CULATR 2311-11), and the procedures were carried out in accordance with the institutional guidelines. e animals were kept under the standardized conditions at the Laboratory Animal Unit of e University of Hong Kong/the Minimal Disease Area with controlled humidity-temperature environment, controlled light-dark regime, sufficient movement allowed, free access to water, and hygienic conditions were provided for the rats
One tissue section is mounted on every tenth slide, and stained to provide a guide map during Laser capture microdissection (LCM) procedures. e first ten and the last ten slides were not used for LCM procedure because the sections were small in size and did not allow reliable identification of the MCC zones. e middle 30–40 slides contained relatively large tissue sections, allowing homogenous isolation of cells from two zones separately using two LCM caps

Summary

Introduction

Articular surfaces of the temporomandibular joint are covered by fibrocartilage, and the one covering the condyle is known as mandibular condylar cartilage (MCC). Mandibular condyle dysfunctions might be associated with temporomandibular joint (TMJ) disorders such as internal derangement, osteoarthrosis, and traumas [1]. 10-11% of the people suffering from temporomandibular joint disorders have symptoms of TMJ-osteoarthrosis [2, 3]. MCC exhibits distinctive biological characteristics as compared with other types of cartilages. MCC plays a dual role: as a growth plate cartilage and as an articular cartilage [4, 5]. MCC is a multizonal heterogeneous fibrocartilage which is composed of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones [1, 6]

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Conclusion