Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Summary

This protocol details the isolation of chondrocytes, Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs), and Migratory Chondroprogenitors (MCPs) from human articular cartilage. It covers enzymatic digestion, fibronectin adhesion, and migration-based assays for isolating and characterizing these cells.

Abstract

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened chondrogenesis, and limited hypertrophic traits. The enrichment of progenitors is achieved through differential fibronectin adhesion and migration-based explant assays, with Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs) and Migratory Chondroprogenitors (MCPs) demonstrating superior potential compared to chondrocytes. This article delves into the details of isolating resident cartilage-derived cells, namely chondrocytes and chondroprogenitors. While valuable insights from chondrocyte research contribute to our understanding of cartilage repair, ongoing efforts are directed toward the use of chondroprogenitors and exploring their potential as an alternative therapeutic approach. Additionally, this methodology article provides a detailed step-by-step protocol for isolating three specific cell types from cartilage: chondrocytes, FAA-CPs, and MCPs. By following standardized procedures, this protocol facilitates the successful extraction of these cell subtypes. Grounded in extensive research, the article focuses on the intricate techniques utilized in isolating the different subsets and the optimized culture conditions required to expand and maintain their cultures. The methodology encompasses enzymatic isolation of human articular cartilage-derived chondrocytes, differential fibronectin adhesion following sequential enzymatic digestion, and migration-based explant assays to obtain cartilage-resident cells.

Introduction

The emergence of cell-based regenerative therapy represents a significant approach to treating cartilage-related ailments, such as osteoarthritis (OA) and chondral defects¹. These disorders, characterized by the breakdown or injury of cartilage within the joints, present substantial challenges during treatment. The self-repair of articular cartilage is reported to be limited due to its aneural architecture, avascularity, and low mitotic activity².

The most utilized cells for cartilage tissue regeneration are Mesenchymal Stem Cells (MSCs) and chondrocytes³. However, seve....

Protocol

The protocol has been approved and complies with the appropriate regulations and guidelines of the Institutional Review Board (Research and Ethics Committee). After obtaining written informed consent, human tibiofemoral joints are procured from osteoarthritis (OA) patients (Kellgren-Lawrence radiological score 4)³⁰ who require total knee replacement as part of their treatment. The joints of patients with any signs of tumors, infections, or inflammatory arthritis (such as rheumatoid arthritis or go.......

Discussion

The potential regeneration of articular cartilage, containing hyaline tissue, may be achieved through the optimization of its two native cell types: chondrocytes and chondroprogenitors. While extensive research on chondrocytes has provided valuable insights into their role in cartilage repair, questions about the nature of the regenerated tissue have prompted efforts to enhance their phenotype and explore alternative therapies³. Chondroprogenitors, identified as a relatively recent cellular subpop.......

Materials

Name	Company	Catalog Number	Comments
22-scalpel blade	GLASS VAN
6-well plate	CORNING	3516
Alcian Blue	THERMO SCIENTIFIC	J6012
Alizarin Red	SIGMA	130223
Amphotericin-B (2 μg/mL).	GIBCO	15240062
Ascorbic acid (62 μg/mL)	SIGMA ALDRICH	A4544-25G
BC CytoFLEX LX flow cytometer	BECKMAN COULTER	CYTExpert Software Version 2.5
CaCl2	SIGMA ALDRICH	C34006
CD105-FITC	BD BIOSCIENCE	561443
CD106-APC	BD BIOSCIENCE	551147
CD14-FITC	BD BIOSCIENCE	555397
CD29-APC	BD BIOSCIENCE	559883
CD34-PE	BD BIOSCIENCE	348057
CD45-FITC	BD BIOSCIENCE	347463
CD49b-FITC	MILTENYL BIOTEC	MACS 130/100337
CD49e-PE	BD BIOSCIENCE	555617
CD73-PE	BD BIOSCIENCE	550257
CD90-PE	BD BIOSCIENCE	561970
Cell counter	DE NOVIX	Cell Drop BF
Cell strainer	HIMEDIA	TCP024
Centrifuge	BECKMAN COULTER	Allegra X-30R
CO2 incubator	THERMO SCIENTIFIC	MODEL-371
Collagen type X (COL10A1),Runt-related transcription factor (RUNX2),SRY-Box Transcription Factor 9 (SOX-9), Aggrecan (ACAN), and Collagen type II (COL2A1)	EUROGENTEC, BELGIUM
Collagenase type II	WORTHINGTON	LS004176
DMEM F-12 (Dulbecco's Modified Eagle's Medium F-12)	SIGMA ALDRICH	D8900-1L
ELISA plate reader	MOLECULAR DEVICES	SpectraMax i3x Reader
Fast Green	FISHER SIENTIFIC	2353459
Fetal Bovine Serum	GIBCO	10270106
FGF2	CLOUD CLONE CORP	APA551Hu01
Fibronectin (10 µL/mL)	SIGMA ALDRICH	F1141
First-Strand synthesis system	TAKARA BIO	6110A
Glutamax	GIBCO	35050061
Hematoxylin	QUALIGENS	Q39411
MgCl2	SIGMA ALDRICH	M8787
Oil Red O	SIGMA	1320065
PBS (Phosphate Buffered Saline)	GIBCO	10010023
PCR thermocycler	APPLIED BIOSYSTEMS	Quantstudio 12K Flex thermocycler
Penicillin-streptomycin (100 IU/mL)	GIBCO	15240062
Picrosirius Red	ALFA AESAR	2610108
Primary antibody (mouse Collagen type II)	DSHB	DSHB II II6B3
Pronase	ROCHE	10165913103
Quant-iT Picogreen dsDNA reagent	THERMO SCIENTIFIC	P7589
Refrigerator	ELANPRO
RNeasy MiniKit	QIAGEN	74104
Safranin O	QUALIGENS	Q39962
Secondary antibody (Goat Anti-Mouse IgG Antibody, HRP conjugate)	THERMO SCIENTIFIC	31430
Shaking water bath	REMI
StemPro differentiating kits	GIBCO	1007201, A1007001, and A1007101
T-25 flask	CORNING	430639
TakyonTM Low Rox SYBR Master Mix dTTP Blue	EUROGENTEC	UF-LSMT-B0701
TGFβ	ABCAM	ab277760
Tissue Culture Petridish	TARSONS	960010
Toluidine Blue	QUALIGENS	2040
Tryphan blue	GIBCO	15250061
Trypsin EDTA	GIBCO	25200072