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, several studies have reported limitations in using these cells for regeneration⁴. These limitations include the terminal differentiation of chondrocytes during extended in vitro expansion to achieve the required cell count and the hypertrophic tendency of MSCs. These factors can result in the formation of repair tissue with a suboptimal combination of fibrocartilage and hyaline^5,6,7,8.

The discovery of Chondroprogenitor cells (CPCs) arose from the quest for alternative cells in articular cartilage⁹. They have generated immense interest due to their resemblance to MSCs and their superior chondrogenic potential, all while exhibiting reduced hypertrophy - an indispensable combination sought after^10,11. In contrast to chondrocytes, these progenitor populations have been reported to exhibit enhanced replicative and telomerase activity, as well as increased expression of Neurogenic locus notch homolog protein 1 (NOTCH-1) and SRY-box transcription factor 9 (SOX-9)^12,13,14,15. There are two standard methods to isolate CPCs reported both from cartilage and meniscus, including one based on their integrin receptor (CD49e/CD29) expression, isolated through a fibronectin adhesion assay - Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs), and the other based on their heightened migratory potential from cartilage explants- Migratory Chondroprogenitors (MCPs)^{11,13,16,17,18,19}. Numerous in vitro studies demonstrate the chondrogenic superiority and reduced hypertrophy of both FAA-CPCs and MCPs compared to chondrocytes and bone marrow (BM)-MSCs^20,21,22,23.

Recent in vitro investigations comparing FAA-CPCs to MCPs have demonstrated the enhanced cartilage regeneration capacity of the latter progenitor population under normal oxygen conditions²⁴. These optimistic in vitro results showcase a hyaline-like regeneration, encouraging further in vivo experiments. Nevertheless, both populations of CPCs have been reported to efficiently repair and regenerate cartilage in OA and other osteochondral disorders in animal models^{25,26,27,28,29}.

Our laboratory has actively contributed to standardizing CPC isolation techniques and comparing their phenotypic characteristics with chondrocytes and MSCs. This article will provide a detailed protocol explaining the steps involved in isolating and culturing cartilage resident cells, namely chondrocytes, FAA-CPCs, and MCPs.

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 gout) were excluded from the study. It is ensured that all procedures are conducted under sterile conditions, adhering to standard laboratory protocols throughout the experimental process.

1. Obtaining tibiofemoral joints

1. Obtain knee joints (tibiofemoral joints) from human donors requiring above-knee amputation as part of treatment or from those with Grade IV osteoarthritis³⁰ (radiological evidence showing marked reduction of joint space with chondral sclerosis) undergoing total knee replacement.
   NOTE: Ensure that informed consent is obtained from patients following the Helsinki Declaration's principles. Ensure adherence to Ethics Committee and Institutional Review Board rules.
2. Exclude any joints showing symptoms of infection or inflammation.

2. Processing harvested joints

1. Place the harvested tibiofemoral joints in 1x phosphate-buffered saline (PBS) solution under sterile conditions.
2. Prepare a sterile area under the hood by placing a sterile underpad. Transfer the harvested joints onto the underpad.
3. Stabilize the sectioned tibiofemoral joints by holding the subchondral bone with the cartilage facing in the upward direction.
4. Using a scalpel blade (no. 22), harvest rectangular-shaped cartilage shavings from the non-weight bearing areas for OA patients within 1-2 h of obtaining the joint.
5. Harvest the cartilage sections of 8 mm x 10 mm from the superficial layer to the deeper layer.
6. Wash the cartilage slices with 1x PBS solution and place them in a Petri dish containing 1-2 mL of plain DMEM medium.
7. Mince the cartilage slices nicely to a size of less than 1 mm x 1mm x 1mm with a scalpel blade (no.22).
   NOTE: Ensure that the cartilage slices or minced cartilage do not dry out; place them in a culture medium or PBS devoid of serum.

3. Isolation of chondrocytes

1. Place the minced cartilage in an upright T-25 flask containing 10 mL of Dulbecco's Modified Eagle Medium F12 (DMEM-F12) with 0.15% collagenase type II for enzymatic digestion. Leave the flask undisturbed for 12-14 h in a CO₂ incubator, ensuring standard culture conditions³¹.
   NOTE: Plain DMEM-F12 medium without traces of serum should be used, as serum inactivates the enzymatic action.
2. Following overnight digestion, transfer the medium containing the released cells into a fresh, sterile centrifuge tube containing an equal volume of DMEM-F12 + 10% Fetal Bovine Serum (FBS), using a cell strainer to separate the cells from the debris. The released cells are the "chondrocytes".
3. Centrifuge the filtered cells at a speed of 1200 x g for 5 mins at 37 °C.
4. Discard the supernatant without disturbing the pellet. Reconstitute the obtained pellet in 1 mL of medium and count the viable cells released using a trypan blue exclusion assay.
5. Load the chondrocytes in a T-25 flask at a concentration of 10,000 cells/cm² and expand to the required passage number using DMEM-F12 containing 10% FBS. Additional components in the medium include ascorbic acid (62 µg/mL), L-glutamine (2.5 mM/L), penicillin-streptomycin (100 IU/mL), and amphotericin-B (2 µg/mL).
6. Refresh the medium every 3 days and harvest cells at sub-confluence using 0.125% Trypsin containing Ethylenediamine tetraacetic acid (EDTA).

4. Isolation of Fibronectin Adhesion Assay-Derived Chondroprogenitors (FAA-CPs)

1. 12 h prior to the release of the chondrocytes, prepare 10 mL of 1x PBS containing 1 mM of MgCl₂ (10 µL) and 1 mM of CaCl₂ (10 µL), and 100 µL of Fibronectin (10 µL/mL)³².
2. Coat the required number of wells of a 6-well plate (1.5 mL/9.3 cm²) using the prepared solution.
3. Seal the plate tightly and refrigerate overnight at 4 °C.
4. Additionally, obtain cartilage shavings in a manner similar to that explained for isolation of chondrocytes (steps 2.1-2.5).
5. Subject the cartilage shavings to sequential overnight enzymatic digestion (0.2% Pronase for 3 h; followed by 0.04% Collagenase type II for 12 h) in a shaking water bath maintained at 37 °C for obtaining individual chondrocytes.
6. The following day, remove the Fibronectin-coated 6-well plate from the refrigerator and remove excess Fibronectin.
   NOTE: Excess Fibronectin should be removed slowly and gently without disturbing the coating on the bottom of the well. The presence of MgCl₂ (10 µL) and 1 mM of CaCl₂ is crucial to ensure the attachment of cells.
7. Add 2-3 mL of plain DMEM-F12 medium into the coated wells.
8. Seed the released chondrocytes onto the coated wells at a loading density of 4000 cells/well and leave the plate undisturbed for a period of 20 min.
9. Post incubation, remove the excess media and non-adherent cells. Add 2-3 mL of standard growth media as used for chondrocytes (DMEM-F12 + 10% FBS).
10. Maintain the adherent cells under standard culture conditions for 10-12 days to obtain CPC clones (colonies of >32 cells).
11. Isolate using 0.125% trypsin-EDTA for 180 s, re-plate the clones at a ratio of 1 clone/5 cm², and expand the enriched polyclonal CPs to the required confluence. These cells obtained are referred to as the "FAA-CPCs".
12. Culture the cells further in DMEM-F12 medium + 10% FBS + transforming growth factor beta 2 (TGFβ2: 1 ng/mL) + fibroblast growth factor (FGF2: 5 ng/mL).
    NOTE: The incubation of cells on fibronectin-coated plates should not exceed 20 min, as chondrocytes may also begin to adhere. During the 20 min adhesion period, the medium must be devoid of serum. Next, the cells that adhere to form clones must be cultured in a medium additionally containing FBS. The clones must be isolated only after ensuring that each clone has more than 32 cells; this is to avoid transit amplifiers. Further expansion of the clones after trypsinization should include the additional growth factors mentioned. A loading density of 4000 chondrocytes/9.3 cm² of a fibronectin-coated plate, following a 20 min incubation followed by a wash, results in the adhesion of 80-100 cells in total. Out of these, around 20 clones will progress to grow and achieve a population doubling of 5 within 10-12 days.

5. Isolation of migratory chondroprogenitors

1. Shave cartilage explants (10 mm x 5 mm x 1 mm ) from the harvested articular joint and place them in a sterile 6-well plate containing DMEM-F12 media with 10% FBS and 10 mM Glutamax (2-3 explants/well)³³.
2. Leave the plate with the explants undisturbed for 48 h in a CO₂ incubator maintained at 37 °C.
3. After 2 days, transfer the explants to a centrifuge tube containing 10 mL of 0.1% collagenase solution for enzymatic digestion. Incubate the tube at 37 °C for 2 h.
   NOTE: The explants require to be rinsed with 1x PBS before enzymatic digestion, as traces of serum can inactivate the enzyme action. For washing, dip the explants in a Petri dish filled with 2-3 mL of 1x PBS; this is also to ensure non-contamination with any released chondrocytes. The handling of the explants must be kept to a minimum, and this is to avoid stressing the progenitors that have started to migrate.
4. Post digestion, rinse the explants with 1x PBS and place them back in the same wells of the plate containing fresh DMEM-F12 media with 10% FBS and 10 mM of Glutamax medium.
5. Maintain the plate in standard culture conditions in the incubator. Observe for the migration of chondroprogenitors in the subsequent days.
6. On reaching sub confluence, harvest the MCPs using 0.125% of Trypsin containing EDTA.
7. Expand the MCPs using the standard expansion medium, which includes DMEM-F12, which contains 10% FBS and 10 mM of Glutamax.

6. Phenotypic characterization of chondrocytes, FAA-CPs and MCPs

1. Flow Cytometric Analysis (FACS)
   NOTE: The following steps are followed for FACS analysis of the harvested cell groups.
   1. Trypsinize the cells as per standard protocol using 0.125% Trypsin and centrifuge at 1200 x g, 5 min, room temperature to obtain the cell pellet.
   2. Discard the supernatant, wash, and re-suspend the pellet with 1x PBS.
   3. Transfer the suspension to labeled tubes for FACS.
   4. Divide the suspension equally into two tubes: an 'unstained' tube that acts as a control (cell suspension without antibody) and 'stained' tubes that act as tests (cell suspension with antibody).
   5. Follow the technical data sheet of individual antibodies/Cluster of differentiation (CD) markers for staining. Antibodies for comparison include CD105-FITC, CD73-PE, CD90-PE, CD106-APC (positive expression markers); CD34-PE, CD45-FITC, and CD14-FITC (negative expression markers)^34,35; CD166-BB515 and CD146-PE (potential chondrogenic markers)^36,37.
      NOTE: CD markers are light-sensitive. To ensure the steps are done in the dark.
   6. Incubate the cell suspension with antibody for 30 min in the dark.
   7. Post staining, add 1 mL of 1x PBS to the tubes and centrifuge at 1200 x g, 5 min, room temperature to obtain the cell pellet.
   8. Discard three-fourths of the supernatant. Re-suspend the pellet in the remaining supernatant content by gentle trituration.
   9. Proceed for FACS analysis.

7. qRT-PCR

NOTE: qRT-PCR analysis of gene expression involves evaluation of Collagen type I (COL1A1), Collagen type X (COL10A1), and Runt-related transcription factor (RUNX2) for hypertrophic expression, and SOX-9, Aggrecan (ACAN), and Collagen type II (COL2A1) for chondrogenesis.

1. Extract RNA from the cell groups using commercially available kits as per the manufacturer's instructions.
2. Post extraction, evaluate the A₂₆₀/A₂₈₀ ratio and the RNA concentration.
3. Utilize 280 ng of the extracted RNA to construct complementary DNA (cDNA).
4. Initiate qRT-PCR using Sybr Green, each reaction containing a final concentration of 7 ng of cDNA on a thermocycler.
5. Normalize each gene's relative mRNA expression to the GAPDH reference housekeeping gene (ΔCt).
6. Calculate the relative expression of each gene using the 2^-ΔΔCt technique by comparing each individual gene's ΔCt value to that of the FAA-CPs (ΔΔCt)³⁷.

8. Multilineage differentiation

NOTE: Commercially available differential media induces trilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages.

1. For adipogenic differentiation, seed cells at a loading density of 1000 cells/cm² in a cell culture plate and culture using adipogenic differentiation medium (medium change - once in 3 days) till a sub confluence of 80% for 3 weeks.
2. For osteogenic and chondrogenic differentiation, follow the 28-day pellet culture system³⁸.
3. Centrifuge 0.5 x 10⁶ cells at 400 x g for 12 min at 37 °C to form pellets and initiate differentiation using osteogenic and chondrogenic differentiation medium.
4. Fix, embed, and section the pellets for confirmatory staining (step 9).

9. Confirmatory staining

1. In the case of adipogenic lineage cells, fix the cells with buffered formalin, wash, and stain with Oil Red O (0.5%). Stain the controls (cultured on a standard DMEM medium with 10% FBS) as well.
2. Observe under a microscope and acquire the images.
3. For the differentiated osteogenic lineage cells, stain with Alizarin Red (2%).
   NOTE: Multiple confirmatory staining protocols are applicable for the chondrogenic differentiated cells.
4. Alcian Blue staining: Stain the fixed cells with Alcian blue for 5 min and counterstain with Neutral Red.
5. For assessing Glycosaminoglycan (GAG) content, perform Safranin O staining: Stain the slides with Wiegert's Iron Hematoxylin followed by subsequent incubation with acid alcohol (1%), fast green solution (0.05%), acetic acid (1%), and Safranin O solution (1%).
6. Toluidine Blue staining: Stain the slides with Toluidine Blue (0.1%) for 5 min.
7. PicroSirius Red staining: Stain the slides with Picrosirius Red (0.1%) and counterstain using Hematoxylin dye.
   NOTE: Following staining, dehydrate the slides using graded alcohol and clear using xylene. Mount slides using Dibutylphthalate Polystyrene Xylene (DPX) mountant, observe them under a microscope, and acquire images.
8. For immunohistochemistry (type II collagen) staining of the chondrogenic differentiated pellet, subject the pellet sections to enzymatic antigen retrieval using the enzymes pronase (1 mg/mL) and hyaluronidase (2.5 mg/mL).
9. Incubate the sections with primary mouse monoclonal anti-collagen type II antibody, followed by 1:250 secondary HRP labeled goat anti-mouse antibody.
10. Stain the slides with 3,3′-Diaminobenzidine (DAB) chromogen and counterstain them using Hematoxylin.
11. Observe under a microscope and capture images.

10. Determining GAG/DNA content

1. Digest the chondrogenic differentiated pellets using papain-cysteine solution at 65 °C for 16 h.
2. Estimate DNA concentration using Picrogreen reagent³⁸ and acquire the fluorescence intensity at wavelengths- excitation: 480 nm, emission: 520 nm using an ELISA plate reader.
3. Evaluate total GAG content using the dimethyl methylene blue dye method³⁸.
4. Measure optical density at 525 nm using an ELISA plate reader.
5. Normalize the GAG values to the DNA values and calculate the total GAG/DNA ratio.

Results

From 86.9 mg of cartilage slices, a chondrocyte cell yield of 1.72 x 10⁵ cells was noted. Upon loading, chondrocytes promptly adhere, presenting an initial rounded cobblestone appearance and transforming into a fibroblastic state upon further expansion (Figure 1 A,B). Seeding these chondrocytes onto fibronectin plates typically results in 2% adhesion, with each cell undergoing clonal growth (Figure 2 C,D), reaching a ...

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