15 ml tube

A 250 μL volume of cell suspension (containing 2.5 × 10⁵ cells, including both living and dead cells) was added to six 15 mL tubes (Falcon) containing a chondrogenic induction medium consisting of Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific), 10 ng/mL transforming growth factor-β3 (TGF-β3, Miltenyi Biotec, Bergisch Gladbach, Germany), 500 ng/mL bone morphogenetic protein 2 (BMP-2, Medtronic, Minneapolis, MN, USA), 40 μg/mL proline (Merck), 100 nM dexamethasone, 100 μg/mL pyruvate (Merck), 1% antibiotic–antimycotic, 50 μg/mL ascorbate-2-phosphate, and 1% ITS + Premix (Becton Dickinson, San Jose, CA, USA). The cells were centrifuged at 450×g for 10 min to form cell pellets, which were cultured for 21 days. The cultured cell pellets were photographed and weighed with a semi micro balance (CPA225D, Sartorius, Gottingen, Germany). The pellets were cut into 5 µm sections and stained with safranin O and toluidine blue.

Synovial MSCs (8 × 10⁵ cells) were suspended in 400 μL medium containing 10% FBS, 95% FBS, or 100% FBS, cryopreserved at − 80 °C for 7 days, and then thawed. A 62.5 μL volume of cell suspension (containing 1.25 × 10⁵ cells, including living and dead cells) was added to six 15 mL tubes (Falcon) containing 0.5 mL chondrogenic induction medium. The cells were pelleted by centrifugation at 450 × g for 10 min and cultured for 21 days (Fig. 1b). The cultured cell pellets were photographed and weighed with a semi micro balance (CPA225D, Sartorius, Gottingen, Germany). The pellets were sectioned and stained with toluidine blue and immunostained with collagen type II (Kyowa Pharma Chemical Co., LTD, Toyama, Japan). The mean and standard deviation were then determined for the pellet weight (4 donors, n = 24).

The MG1655 strain was transformed with a low-copy-number plasmid (SC101 origin) carrying the P_hiuH promoter sequence upstream of the coding sequence for gfpmut2 (green fluorescent protein) (13 (link)). Cells were grown in either 15-mL tubes (Falcon) or 50-mL flasks (Erlenmeyer) overnight (approximately 14 h) using different volumes of growth media (0.5, 1, and 5 mL for growth in tubes and 5, 10, and 20 mL for growth in flasks) supplemented or not with 0.4% BD Bacto CASA. One microliter of cells was collected, spotted onto 1% agarose (Bio-Rad no. 1613100) pads, and imaged at ×100 (numerical aperture [NA] 1.45 objective) using a Nikon Ti-E inverted fluorescence microscope equipped with a complementary metal oxide semiconductor (CMOS) camera (Hamamatsu Orca Fusion). Single-cell outlines were automatically segmented from phase-contrast images using a modified version of MicrobeTracker (24 (link)) combined with SuperSegger (25 (link)). The P_hiuH-GFP intensity per cell was measured from the average pixel intensity within each segmented cell using a custom-written MATLAB (MathWorks) script. For each replicate, the coefficient of variation was calculated as σ/μ, where σ is the standard deviation and μ is the mean of the single-cell fluorescence level in the population.

Genomic DNA (gDNA) was extracted from all cell lines using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions. cfDNA was recovered either from: (i) 3 mL cell conditioned medium: 4 × 10⁵ cells were seeded in a T25 flask (Corning), once culture reached 80% confluency, the medium was renewed. Then, after 12 h it was collected in 15 mL tubes (Falcon) and spun at 300 g for 5 min to eliminate contaminating cells and cell debris. (ii) 2 mL plasma/serum samples, or (iii) 100–300 μL aqueous humor samples using the QIAamp Circulating Nucleic Acid (CNA) Kit (QIAGEN). Isolated DNA was stored in AVE buffer (RNase-free water with 0.04% sodium azide-QIAGEN) in a 25 uL final volume and quantified using Qubit 2.0 fluorometer with Qubit dsDNA HS assay reagents (Supplementary Figure 2A-E and 3A) (Thermofisher Scientific).

Macropellets were formed in 15-mL tubes (BD Falcon) using the conventional method. MSC macropellets contained only 2.2 x 10⁵ MSCs in 1mL chondrogenic media in tubes. MSC+CD macropellets contained both 2.2 x 10⁵ MSCs in 1 mL chondrogenic media and 1 mg CD, described in previous section, in tubes. CD macropellets contained only 1 mg CD in 1 mL chondrogenic media in tubes. Then the tubes were centrifuged at 400xg for 5 minutes. The macropellets were cultured in a 2% O₂ and 5% CO₂ incubator at 37°C for 14 days with loosened lids to enable gas exchange.

The 4T1 cells were cultured in the presence of 8 μg/mL TRF in T25 flasks overnight at 37 °C in a humidified incubator with 5% CO₂ (Heraeus, Germany). The next day the 4T1 cells were collected into a 15-mL tube (Falcon, Atlanta, GA, USA) and harvested by centrifugation (1000 rpm for 5 min). The supernatant was discarded and the cells were resuspended in 1 mL culture medium. Tumor lysate (TL) from the 4T1 cells was prepared by subjecting these cells to several freeze–thaw cycles. The cells were first frozen in liquid nitrogen and then rapidly thawed at 65 °C. The freeze–thaw cycle was repeated 3–5 times. The cell lysates was centrifuged (2000 rpm for 5 min) and the supernatant was passed through a 30-μm nylon filter-column before it was aliquoted in vials and stored at −80 °C until use.