Safety and efficacy of human juvenile chondrocyte-derived cell sheets for osteochondral defect treatment

Cartilage sampling from juvenile human polydactyly donors

Cartilage from the phalanx and metacarpal bones of amputated polydactylous fingers and toes from 12 patients (24.8 ± 17.0 months old) (Table 1) was sharply dissected using a scalpel and maintained in saline immediately following harvest. All patients were prospectively enrolled at Intermountain Primary Children’s Hospital (Salt Lake City, USA). Institutional Review Board oversight from University of Utah and Intermountain Primary Children’s Hospital was waived due to use of deidentified routine surgical discards.

Chondrocyte isolation

Cartilage harvested from juvenile donor tissues was transferred into saline, cut into <4 mm² pieces by scalpel, and then incubated with 5 mg/mL of Type 1 collagenase at 37 °C for 1.5–3.0 h (LS004197, Worthington Biochemical, Lakewood, USA). Resulting cells were filtered through a 100-µm cell strainer, washed with saline and then resuspended in chondrocyte culture medium (DMEM-F12, 11320082, ThermoFisher Scientific, Waltham, USA) containing 1% antibiotic-antimycotic (15240062, ThermoFisher) and 20% fetal bovine serum (FBS) (16000044, ThermoFisher).

Cell culture

Isolated chondrocytes were seeded on polystyrene dishes (CELLTREAT, Pepperell, USA) at 5000–10,000 cells/cm² in a chondrocyte culture medium (described above). The medium was replaced with chondrocyte medium supplemented with 100 μg/mL L-ascorbic acid phosphate magnesium salt n-hydrate (013–19641, Fujifilm Wako Pure Chemical, Osaka, Japan) at the first medium change on day 4. Cells were passaged with this medium thereafter with the daily observation by phase-contrast microscopy throughout cell culture. Subconfluent cells were collected by TrypLE Select (12563011, ThermoFisher) dissociation and counted. Expanded cells were cryopreserved in STEM-CELLBANKER GMP grade (Zenoaq, Fukushima, Japan) at the end of P0. Serial subculture was performed with the thawed cells at the initial density of 10,000 cells/cm² passaged every 3–5 days.

Juvenile chondrocyte sheet preparation

Cell sheets were prepared from passage 1 cells sourced from thawed cryopreserved cells. Subconfluent P1 cells were collected with 1x TrypLE Select for 5 min, then seeded at a density of 10,000 cells/cm² on temperature-responsive cell culture inserts (CellSeed, Tokyo, Japan). Chondrocyte culture media was changed every 3–4 days. After 2 weeks of culture, cell sheets were harvested with forceps after incubation at room temperature.

Cell viability and total cell number of chondrocyte sheet

Single cells from fabricated JCC sheets were isolated by incubation with TrypLE Select for 15 min and 0.25 mg/mL collagenase P (11 213 857 001, Roche, Basel, Switzerland) for 30 min. Cells were counted via hemocytometer and cell viability was demonstrated via trypan blue (T8154, MilliporeSigma) dye exclusion⁵⁶.

Chondrogenic differentiation culture

Chondrogenic pellet culture was performed based on a previous study³⁸. JCCs harvested at the end of P2 and P9 cultures or isolated cells from P2 JCC sheets were aliquoted in chondrocyte culture medium at 2.5 × 10⁵ cells in 15 mL conical tubes for pellet cultures. Tubes were spun at 500 × g for 10 min. Caps were loosened and cells were incubated at 37 °C, 5% CO₂ for 3 days to facilitate pellet formation. After the 3-day incubation step, chondrogenic samples were induced with chondrogenic medium, control samples were replaced with a new chondrocyte culture medium, and all samples were transferred to a hypoxic incubator (37 °C, 5% CO₂, 5% O₂). Chondrogenic medium contained high glucose DMEM supplemented with 10 ng/mL transforming growth factor beta-3 (TGFβ3) (ThermoFisher), 200 ng/mL bone morphogenic protein-6 (BMP6) (PeproTech), 1% Insulin-Transferrin-Selenium (ITS-G) (ThermoFisher), 1% PS (Life Technologies), 1% non-essential amino acids (NEAA) (ThermoFisher), 100 nM dexamethasone (MP Biomedicals, Irvine, USA), 1.25 mg/ml bovine serum albumin (BSA) (MilliporeSigma), 50 μg/mL L-ascorbic acid phosphate magnesium salt n-hydrate (Fujifilm Wako Pure Chemical), 40 μg/mL L-proline (MilliporeSigma), and 5.35 μg/mL linoleic acid (MilliporeSigma). Media was changed twice a week for 3 weeks.

In vitro cell transformation assay

In vitro cell transformation was evaluated by serial passage culture and soft agar assay. Juvenile chondrocytes were passaged up to 13 passages (57–67 days in total) with a seeding density of 10,000 cells/cm². Cells were observed every day and cell growth rate was calculated as doubling time. Cell transformation was assessed by detecting anchorage-free proliferation. Cultured cells isolated from P2 chondrocyte sheets of normal culture periods (2 weeks) and extended culture periods (3.5 weeks) were seeded in a soft agar gel with chondrocyte culture media with ascorbate at a density of 5,000 cells per well by using CytoSelect Cell Transformation Assay (CBA-140, Cell Biolabs, San Diego, USA). Fluorescent signal representing cell number was measured with a spectrofluorometer (Cytation 3 image reader, BioTek, Winooski, USA) on day 0 and day 8 according to manufacturer’s protocol. HepG2 cells (HB-8065, ATCC) in DMEM containing 10% FBS and 1% antibiotic-antimycotic were used as positive control for anchorage-free cell growth. Data are shown as averages of relative fluorescent units from duplicate or triplicate assays.

Flow cytometry

Isolated cell suspensions from chondrocyte sheets (dissociated as described before) were aliquoted and incubated in 1 µg/mL Fc block solution (564220, BD, Franklin Lakes, USA), resuspended in 10% FBS-containing PBS for 5–10 min, then labeled with fluorescent-conjugated antibodies (Supplementary Table 1) for 15 min with brief vortexing steps. Cells were washed with 10% FBS-containing PBS, centrifuged, resuspended with 1:1000 propidium iodide (PI) (556463, BD) in 10% FBS-containing PBS. Samples were analyzed with a cell analyzer (Canto, BD). Doublets were excluded with FSC-W and SSC-W gating, then the PI-negative population was analyzed for specific antibody staining. The gating strategy is shown in Supplementary Fig. 1.

Histology

Fabricated cell sheets were fixed with 4% paraformaldehyde for 30 min at RT. Harvested rat knee tissue was fixed in 4% paraformaldehyde for four days and decalcified in RapidCal Immuno (BBC Biochemical, Mount Vernon, USA) for one day at RT. Samples were embedded in paraffin blocks and then cut into 5-µm transverse sections with a microtome. Slides were deparaffinized by baking in an oven at 65 °C and subsequent washes with xylene and ethanol. Sections were hydrated by gradual ethanol replacement by distilled water. Safranin-O was used for metachromatic staining for sulfated glycosaminoglycans. Samples were stained for 5 min with Wiegert’s Iron Hematoxylin (MilliporeSigma), 5 min with 0.5 g/L Fast Green FCF (MilliporeSigma), and 5 min with 0.1% Safranin-O (MilliporeSigma). Images were taken with a BX41 microscope (Olympus, Tokyo, Japan) and AmScope Software (USA).

Immunohistochemistry

Sections of histology samples were hydrated, then antigen retrieval was performed. The retrieval method was chosen to preserve the tissue integrity of knee samples and cell sheet samples after the staining optimization: protease K (S3020, Agilent Technologies, Santa Clara, USA) for COL2 and vimentin staining of knee samples; heat antigen retrieval in citrate buffer (pH 6.0) (C9999, MilliporeSigma, Burlington, USA) for COL2 staining of cell sheet samples. Peroxidase blocking was performed with Hydrogen Peroxide Blocking Reagent (ab64218, Abcam, Cambridge, UK). After blocking with 5% donkey serum and 0.1% Triton-X in PBS for 1 h. Samples were then incubated overnight with primary antibodies at 4 °C. Polyclonal goat anti-type I collagen (1:200, SouthernBiotech, Birmingham, USA), monoclonal mouse anti-type II collagen (1:200, 2B1.5, ThermoFisher), polyclonal goat anti-aggrecan (1:100, AF1220, R&D Systems, Minneapolis, USA), and monoclonal rabbit anti-human vimentin (1:200, SP20, Abcam)^57,58 were used as primary antibodies. Normal mouse IgG_2a (X0943, Agilent), normal goat IgG (NI02, MilliporeSigma), or normal rabbit IgG (X0903, Agilent) were used as isotype controls at the same concentration as the primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:1,000, 115–035–166, Jackson ImmunoResearch, West Grove, USA) was used for type II collagen. HRP-conjugated donkey anti-goat antibody (1:1,000, 705-035-147, Jackson) was used for type I collagen and aggrecan staining. HRP-conjugated goat anti-rabbit antibody (1:1,000, 111-035-144, Jackson) was used for human vimentin staining. ImmPACT DAB Peroxidase (HRP) Substrate (SK-4105, Vector Laboratories, Burlingame, USA) was used as a chromogen. Brightfield images were taken with a BX41 microscope and AmScope Software. Fluorescent images were taken with Axio Vert.A1 microscope and ZEN software (Zeiss, Oberkochen, Germany). All primary and secondary antibodies are listed in Supplementary Tables 2 and 3, respectively.

Surgical and transplantation procedure

The animal study plan was evaluated and approved by Institutional Animal Care & Use Committee (IACUC, University of Utah) (assigned ID: 17-09011). SD rats and nude rats (6-week-old), male and female, were purchased from Charles River Laboratories (Wilmington, USA). After a week of acclimatization at the animal facility, animals were anesthetized using isoflurane and O₂ gas. A medial parapatellar incision was made to expose the knee joint; the patella was laterally dislocated and a focal osteochondral defect (diameter 2 mm; depth 200–350 μm) was created on the patellar groove of the femur using an electric grinder without penetration to the bone marrow. Defect depth was controlled by the procedure under a surgical stereo zoom microscope (SZX10, Olympus, Japan) with repeated depth measurement with a needle tip (25 G, BD). Chondrocyte sheets prepared in temperature-responsive cell culture inserts in six-well plates (described above) were washed with saline, then cut in halves by a razor and single sheet halves transplanted to each surgical knee defect after defect creation. All animals received buprenorphine for 2 days and carprofen for 3 days in compliance with the IACUC protocol. Animals were sacrificed after 4, 8, 12, and 24 weeks for further histological evaluations.

Rat hind limb weight-bearing distribution assessment

Weight distribution in rats was demonstrated with Incapacitance Tester (Linton Instrumentation, UK), a device with two separate boards on which the rats sit, measuring how the rats distribute their weight. All animals were acclimatized 1–2 times before and after surgery. No measurement was done until 2-week time point after surgery to avoid effects of muscle trauma and analgesia. Weight distribution was calculated by the following formula:

Sample numbers at week 2, 3, 4, 5, and 6 = 13, 12, 12, 8, and 8 (nontreatment group); = 16, 15, 15, 9, and 9 (defect + CS group), respectively. Data are shown as mean and SEM. **p < 0.01, *p < 0.05 by Welch’s t test.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.