We would like to acknowledge Ms. Bhavini Krishnan and Ms.Merin Mary Zachariah for their intellectual input and the Centre for Stem Cell Research (A unit of inStem Bengaluru), Department of Physiology, Christian Medical College, Vellore, for infrastructural support. The ongoing projects are supported by the Department of Biotechnology (BT/PR32777/MED/31/415/2019), Govt. of India, Science and Engineering Research Board (CRG/2022/004277), Govt. of India, and Fluid Research Grants, Christian Medical College, Vellore.

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
<ol>
	<li>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&#39;s principles. Ensure adherence to Ethics Committee and Institutional Review Board rules.</li>
	<li>Exclude any joints showing symptoms of infection or inflammation.</li>
</ol>
2. Processing harvested joints
<ol>
	<li>Place the harvested tibiofemoral joints in 1x phosphate-buffered saline (PBS) solution under sterile conditions.</li>
	<li>Prepare a sterile area under the hood by placing a sterile underpad. Transfer the harvested joints onto the underpad.</li>
	<li>Stabilize the sectioned tibiofemoral joints by holding the subchondral bone with the cartilage facing in the upward direction.</li>
	<li>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.</li>
	<li>Harvest the cartilage sections of 8 mm x 10 mm from the superficial layer to the deeper layer.</li>
	<li>Wash the cartilage slices with 1x PBS solution and place them in a Petri dish containing 1-2 mL of plain DMEM medium.</li>
	<li>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.</li>
</ol>
3. Isolation of chondrocytes
<ol>
	<li>Place the minced cartilage in an upright T-25 flask containing 10 mL of Dulbecco&#39;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.</li>
	<li>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 &#34;chondrocytes&#34;.</li>
	<li>Centrifuge the filtered cells at a speed of 1200 x g for 5 mins at 37 &#176;C.</li>
	<li>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.</li>
	<li>Load the chondrocytes in a T-25 flask at a concentration of 10,000 cells/cm&#178; and expand to the required passage number using DMEM-F12 containing 10% FBS. Additional components in the medium include ascorbic acid (62 &#181;g/mL), L-glutamine (2.5 mM/L), penicillin-streptomycin (100 IU/mL), and amphotericin-B (2 &#181;g/mL).</li>
	<li>Refresh the medium every 3 days and harvest cells at sub-confluence using 0.125% Trypsin containing Ethylenediamine tetraacetic acid (EDTA).</li>
</ol>
4. Isolation of Fibronectin Adhesion Assay-Derived Chondroprogenitors (FAA-CPs)
<ol>
	<li>12 h prior to the release of the chondrocytes, prepare 10 mL of 1x PBS containing 1 mM of MgCl2 (10 &#181;L) and 1 mM of CaCl2 (10 &#181;L), and 100 &#181;L of Fibronectin (10 &#181;L/mL)32.</li>
	<li>Coat the required number of wells of a 6-well plate (1.5 mL/9.3 cm2) using the prepared solution.</li>
	<li>Seal the plate tightly and refrigerate overnight at 4 &#176;C.</li>
	<li>Additionally, obtain cartilage shavings in a manner similar to that explained for isolation of chondrocytes (steps 2.1-2.5).</li>
	<li>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 &#176;C for obtaining individual chondrocytes.</li>
	<li>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 &#181;L) and 1 mM of CaCl2 is crucial to ensure the attachment of cells.</li>
	<li>Add 2-3 mL of plain DMEM-F12 medium into the coated wells.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Maintain the adherent cells under standard culture conditions for 10-12 days to obtain CPC clones (colonies of &#62;32 cells).</li>
	<li>Isolate using 0.125% trypsin-EDTA for 180 s, re-plate the clones at a ratio of 1 clone/5 cm&#178;, and expand the enriched polyclonal CPs to the required confluence. These cells obtained are referred to as the &#34;FAA-CPCs&#34;.</li>
	<li>Culture the cells further in DMEM-F12 medium + 10% FBS + transforming growth factor beta 2 (TGF&#946;2: 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.</li>
</ol>
5. Isolation of migratory chondroprogenitors
<ol>
	<li>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.</li>
	<li>Leave the plate with the explants undisturbed for 48 h in a CO2 incubator maintained at 37 &#176;C.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
	<li>Maintain the plate in standard culture conditions in the incubator. Observe for the migration of chondroprogenitors in the subsequent days.</li>
	<li>On reaching sub confluence, harvest the MCPs using 0.125% of Trypsin containing EDTA.</li>
	<li>Expand the MCPs using the standard expansion medium, which includes DMEM-F12, which contains 10% FBS and 10 mM of Glutamax.</li>
</ol>
6. Phenotypic characterization of chondrocytes, FAA-CPs and MCPs
<ol>
	<li>Flow Cytometric Analysis (FACS) 
	NOTE: The following steps are followed for FACS analysis of the harvested cell groups.
	<ol>
		<li>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.</li>
		<li>Discard the supernatant, wash, and re-suspend the pellet with 1x PBS.</li>
		<li>Transfer the suspension to labeled tubes for FACS.</li>
		<li>Divide the suspension equally into two tubes: an &#39;unstained&#39; tube that acts as a control (cell suspension without antibody) and &#39;stained&#39; tubes that act as tests (cell suspension with antibody).</li>
		<li>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.</li>
		<li>Incubate the cell suspension with antibody for 30 min in the dark.</li>
		<li>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.</li>
		<li>Discard three-fourths of the supernatant. Re-suspend the pellet in the remaining supernatant content by gentle trituration.</li>
		<li>Proceed for FACS analysis.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Extract RNA from the cell groups using commercially available kits as per the manufacturer&#39;s instructions.</li>
	<li>Post extraction, evaluate the A260/A280 ratio and the RNA concentration.</li>
	<li>Utilize 280 ng of the extracted RNA to construct complementary DNA (cDNA).</li>
	<li>Initiate qRT-PCR using Sybr Green, each reaction containing a final concentration of 7 ng of cDNA on a thermocycler.</li>
	<li>Normalize each gene&#39;s relative mRNA expression to the GAPDH reference housekeeping gene (&#916;Ct).</li>
	<li>Calculate the relative expression of each gene using the 2-&#916;&#916;Ct technique by comparing each individual gene&#39;s &#916;Ct value to that of the FAA-CPs (&#916;&#916;Ct)37.</li>
</ol>
8. Multilineage differentiation
NOTE: Commercially available differential media induces trilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages.
<ol>
	<li>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.</li>
	<li>For osteogenic and chondrogenic differentiation, follow the 28-day pellet culture system38.</li>
	<li>Centrifuge 0.5 x 106 cells at 400 x g for 12 min at 37 &#176;C to form pellets and initiate differentiation using osteogenic and chondrogenic differentiation medium.</li>
	<li>Fix, embed, and section the pellets for confirmatory staining (step 9).</li>
</ol>
9. Confirmatory staining
<ol>
	<li>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.</li>
	<li>Observe under a microscope and acquire the images.</li>
	<li>For the differentiated osteogenic lineage cells, stain with Alizarin Red (2%). 
	NOTE: Multiple confirmatory staining protocols are applicable for the chondrogenic differentiated cells.</li>
	<li>Alcian Blue staining: Stain the fixed cells with Alcian blue for 5 min and counterstain with Neutral Red.</li>
	<li>For assessing Glycosaminoglycan (GAG) content, perform Safranin O staining: Stain the slides with Wiegert&#39;s Iron Hematoxylin followed by subsequent incubation with acid alcohol (1%), fast green solution (0.05%), acetic acid (1%), and Safranin O solution (1%).</li>
	<li>Toluidine Blue staining: Stain the slides with Toluidine Blue (0.1%) for 5 min.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Incubate the sections with primary mouse monoclonal anti-collagen type II antibody, followed by 1:250 secondary HRP labeled goat anti-mouse antibody.</li>
	<li>Stain the slides with 3,3&#8242;-Diaminobenzidine (DAB) chromogen and counterstain them using Hematoxylin.</li>
	<li>Observe under a microscope and capture images.</li>
</ol>
10. Determining GAG/DNA content
<ol>
	<li>Digest the chondrogenic differentiated pellets using papain-cysteine solution at 65 &#176;C for 16 h.</li>
	<li>Estimate DNA concentration using Picrogreen reagent38 and acquire the fluorescence intensity at wavelengths- excitation: 480 nm, emission: 520 nm using an ELISA plate reader.</li>
	<li>Evaluate total GAG content using the dimethyl methylene blue dye method38.</li>
	<li>Measure optical density at 525 nm using an ELISA plate reader.</li>
	<li>Normalize the GAG values to the DNA values and calculate the total GAG/DNA ratio.</li>
</ol>

Chondroprogenitor and Chondrocyte Isolation from Human Articular Cartilage

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...

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 hyaline5,6,7,8.
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 after10,11. 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)-MSCs20,21,22,23.
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 models25,26,27,28,29.
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.

<table><tbody><tr><td>22-scalpel blade&amp;nbsp;</td><td>GLASS VAN</td><td></td><td></td></tr><tr><td>6-well plate&amp;nbsp;</td><td>CORNING</td><td>3516</td><td></td></tr><tr><td>Alcian Blue</td><td>THERMO SCIENTIFIC</td><td>J6012</td><td></td></tr><tr><td>Alizarin Red&amp;nbsp;</td><td>SIGMA</td><td>130223</td><td></td></tr><tr><td>Amphotericin-B (2 &amp;mu;g/mL).</td><td>GIBCO</td><td>15240062</td><td></td></tr><tr><td>Ascorbic acid (62 &amp;mu;g/mL)</td><td>SIGMA ALDRICH</td><td>A4544-25G</td><td></td></tr><tr><td>BC CytoFLEX LX flow cytometer&amp;nbsp;</td><td>BECKMAN COULTER</td><td>CYTExpert Software Version 2.5</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA ALDRICH</td><td>&amp;nbsp;C34006</td><td></td></tr><tr><td>CD105-FITC</td><td>BD BIOSCIENCE</td><td>561443</td><td></td></tr><tr><td>CD106-APC</td><td>BD BIOSCIENCE</td><td>551147</td><td></td></tr><tr><td>CD14-FITC</td><td>BD BIOSCIENCE</td><td>555397</td><td></td></tr><tr><td>CD29-APC</td><td>BD BIOSCIENCE</td><td>559883</td><td></td></tr><tr><td>CD34-PE</td><td>BD BIOSCIENCE</td><td>348057</td><td></td></tr><tr><td>CD45-FITC</td><td>BD BIOSCIENCE</td><td>347463</td><td></td></tr><tr><td>CD49b-FITC</td><td>MILTENYL BIOTEC</td><td>&amp;nbsp;MACS 130/100337</td><td></td></tr><tr><td>CD49e-PE</td><td>BD BIOSCIENCE</td><td>555617</td><td></td></tr><tr><td>CD73-PE</td><td>BD BIOSCIENCE</td><td>550257</td><td></td></tr><tr><td>CD90-PE</td><td>BD BIOSCIENCE</td><td>561970</td><td></td></tr><tr><td>Cell counter</td><td>DE NOVIX</td><td>Cell Drop BF</td><td></td></tr><tr><td>Cell strainer</td><td>HIMEDIA</td><td>TCP024</td><td></td></tr><tr><td>Centrifuge</td><td>BECKMAN COULTER</td><td>Allegra X-30R</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator&amp;nbsp;</td><td>THERMO SCIENTIFIC</td><td>MODEL-371</td><td></td></tr><tr><td>Collagen type X (COL10A1),Runt-related transcription factor (RUNX2),SRY-Box Transcription Factor 9 (SOX-9), Aggrecan (ACAN), and Collagen type II (COL2A1)&amp;nbsp;</td><td>EUROGENTEC, BELGIUM</td><td></td><td></td></tr><tr><td>Collagenase type II</td><td>WORTHINGTON</td><td>LS004176</td><td></td></tr><tr><td>DMEM F-12 (Dulbecco&#039;s Modified Eagle&#039;s Medium F-12)</td><td>SIGMA ALDRICH</td><td>D8900-1L</td><td></td></tr><tr><td>ELISA plate reader</td><td>MOLECULAR DEVICES</td><td>SpectraMax i3x Reader</td><td></td></tr><tr><td>Fast Green</td><td>FISHER SIENTIFIC</td><td>2353459</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO</td><td>10270106</td><td></td></tr><tr><td>FGF2</td><td>CLOUD CLONE CORP</td><td>APA551Hu01</td><td></td></tr><tr><td>Fibronectin (10 &amp;micro;L/mL)</td><td>SIGMA ALDRICH</td><td>F1141</td><td></td></tr><tr><td>First-Strand synthesis system</td><td>TAKARA BIO</td><td>6110A</td><td></td></tr><tr><td>Glutamax</td><td>GIBCO</td><td>35050061</td><td></td></tr><tr><td>Hematoxylin</td><td>QUALIGENS</td><td>Q39411</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>SIGMA ALDRICH</td><td>M8787</td><td></td></tr><tr><td>Oil Red O</td><td>SIGMA</td><td>1320065</td><td></td></tr><tr><td>PBS (Phosphate Buffered Saline)</td><td>GIBCO</td><td>10010023</td><td></td></tr><tr><td>PCR thermocycler&amp;nbsp;</td><td>APPLIED BIOSYSTEMS</td><td>Quantstudio 12K Flex thermocycler&amp;nbsp;</td><td></td></tr><tr><td>Penicillin-streptomycin (100 IU/mL)</td><td>GIBCO</td><td>15240062</td><td></td></tr><tr><td>Picrosirius Red</td><td>ALFA AESAR</td><td>2610108</td><td></td></tr><tr><td>Primary antibody (mouse Collagen type II)</td><td>DSHB</td><td>DSHB II II6B3</td><td></td></tr><tr><td>Pronase</td><td>ROCHE</td><td>10165913103</td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA reagent</td><td>THERMO SCIENTIFIC</td><td>P7589</td><td></td></tr><tr><td>Refrigerator</td><td>ELANPRO</td><td></td><td></td></tr><tr><td>RNeasy MiniKit</td><td>QIAGEN</td><td>74104</td><td></td></tr><tr><td>Safranin O</td><td>QUALIGENS</td><td>Q39962</td><td></td></tr><tr><td>Secondary antibody (Goat Anti-Mouse IgG Antibody, HRP conjugate)</td><td>THERMO SCIENTIFIC</td><td>31430</td><td></td></tr><tr><td>Shaking water bath&amp;nbsp;</td><td>REMI</td><td></td><td></td></tr><tr><td>StemPro differentiating kits&amp;nbsp;</td><td>GIBCO</td><td>1007201, A1007001, and A1007101</td><td></td></tr><tr><td>T-25 flask&amp;nbsp;</td><td>CORNING</td><td>430639</td><td></td></tr><tr><td>TakyonTM Low Rox SYBR Master Mix dTTP Blue&amp;nbsp;</td><td>EUROGENTEC</td><td>UF-LSMT-B0701</td><td></td></tr><tr><td>TGF&amp;beta;</td><td>ABCAM</td><td>ab277760</td><td></td></tr><tr><td>Tissue Culture Petridish</td><td>TARSONS</td><td>960010</td><td></td></tr><tr><td>Toluidine Blue</td><td>QUALIGENS</td><td>2040</td><td></td></tr><tr><td>Tryphan blue&amp;nbsp;</td><td>GIBCO</td><td>15250061</td><td></td></tr><tr><td>Trypsin EDTA</td><td>GIBCO</td><td>25200072</td><td></td></tr></tbody></table>

isolation of chondrocytes and chondroprogenitors using fibronectin adhesion and migratory assay

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.

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 ...

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

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.

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened ...

isolation-chondrocytes-chondroprogenitors-using-fibronectin-adhesion

Research

JoVE Journal

Biology

470 Views.  Christian Medical College. Op het gebied van kraakbeenweefselmanipulatie en regeneratieve geneeskunde is er nog steeds behoefte aan het verbeteren van biologische en functionele resultaten in termen van het verbeteren van lopende behandelingen en het ontwikkelen van nieuwe therapeutische strategieën. Huidig preklinisch onderzoek wijst op het mogelijke gebruik van van kraakbeen afgeleide chondroprogenitors als een haalbare optie voor kraakbeengenezing. Huidig onderzoek omvat strategie?...

Video: Isolatie van chondrocyten en chondroprogenitoren met behulp van fibronectine-adhesie en migratoire assay

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Developmental Biology

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Bioengineering

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

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