Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering

Abstract

Objective The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. Design Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity Results Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. Conclusions The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

Isolation of High‐Quality Total RNA from Small Animal Articular Cartilage for Next‐Generation Sequencing

“…This is mainly attributable to the dense extracellular matrix (ECM) of articular cartilage, which prevents compounds conventionally used to lyse cells and extract nucleic acids (such as TRIzol) from accessing tissue‐resident chondrocytes and cartilage progenitor cells. As a result, cartilage must be pre‐processed before RNA extraction using either harsh homogenization conditions (Le Bleu et al., 2017; Ruettger, Neumann, Wiederanders, & Huber, 2010) or enzymatic digestion methods that isolate chondrocytes from articular cartilage tissues (Lau, Peck, Huang, & Wang, 2015; Yan et al., 2021; Yonenaga et al., 2017). However, homogenization protocols compromise RNA integrity (Ruettger et al., 2010), and the lengthy cell isolation procedures during enzymatic digestion of cartilage ECM may introduce changes in the transcriptome of chondrocytes, which decreases the suitability of such protocols for efficient application of high‐sensitivity downstream applications when studying normal cartilage homeostasis.…”