Purpose

As an advanced therapy medicinal product (ATMP), autologous chondrocyte implantation (ACI) is subject to strict regulation, for example concerning the fate and biodistribution of the implant in a possible case of delamination. The present study assessed the stability of fluorescence labelling of chondrocyte spheroids with PKH26 in matrix-associated ACI and its applicability to recover the spheroids after a simulated biodistribution in the ovine stifle joint.

Methods and Materials

Ovine chondrocytes were labelled with PKH26 dye (Sigma-Aldrich) and cultured for 6 weeks in 3D environment for spheroid formation and general evaluation. After one week, 6 labelled spheroids were injected into the intact stifle joint capsule of merino sheep (n=3). After 9 days, the capsule of euthanised animals was opened to trace the labelled spheroids using a CO₂-laser pointer (YAG 532 nm) and a long pass filter (EM LP590) or laser protection glasses (KTP/YAG LGF 532 nm) for detection. Detected spheroids were dissected and formalin-fixed for histological verification by haematoxylin-eosin (HE) staining.

Results

Labelling was stable in vitro for 6 weeks (Figure 1) and showed no impact on the spheroids’ common characteristics. PKH26-labelled spheroids appeared pink in visible light and red or yellow in fluorescent light, depending on the detection system (Figure 2). In vivo, all injected spheroids were recovered from different locations close to the injection site in the stifle joint capsule. HE staining confirmed the spheroids´ localisation in the connective tissue.

Conclusion

Fluorescence labelling was successfully used for visualisation, detection, and recovery of ectopically located chondrocyte spheroids in the ovine stifle joint. This technique may thus be suitable to recover delaminated or delocalised cell transplants and, in addition, to track down labelled chondrocyte spheroids in biodistribution studies.

Purpose

Spheroids of human autologous matrix-associated chondrocytes are clinically available for autologous chondrocyte implantation (ACI). The cartilage cell implant attaches to the defect within 20 minutes without any further fixation. However, the mode of action of this adhesion process has not been elucidated yet. The aim of this study was to identify specific adhesion molecules involved in spheroid adhesion to the subchondral cartilage defect.

Methods and Materials

Chondrocytes were isolated from 8 donors (m/f 1:1, 54-76 years). Then, spheroids were generated, cultured for 2 weeks and implanted in an ex vivo human subchondral cartilage defect model [1] (70 spheroids/cm²) for adhesion experiments. At different time points after implantation (0, 10 and 20 minutes, 1, 4 and 24 hours), samples were taken for gene expression analysis of adhesion molecules CD44, dermatopontin, desmocollin 2, fibulin 7, integrin alpha 1, 2, 10 and 11, integrin beta 3 and transmembrane protein 204 using quantitative RT-PCR. Protein expression of CD44, integrin alpha 2 and integrin beta 3 was analysed immunohistochemically.

[1] C Bartz, J Transl Med. 2016 Nov 15;14(1):317. doi: 10.1186/s12967-016-1065-8.

Results

Implanted spheroids adhered to the defect ground, defect borders and to each other within 10 minutes. Within 24 hours gene expression of all adhesion molecules was upregulated in implanted spheroids compared to non-implanted spheroids (Figure 1). On protein level, CD44 and integrin alpha 2, but no integrin beta 3 expression was detected in both non-implanted spheroids and after implantation (Figure 2). Adhesion sites showed no expression of integrin alpha 2 and integrin beta 3. CD44 was detected in cells involved at the different adhesion sites and between implanted spheroids.

Conclusion

The gene expression profile of different adhesion molecules in the cartilage cell implant was changed after implantation. CD44 played a major role during adhesion, whereas integrin alpha 2 and integrin beta 3 were not involved.

Purpose

In clinical practice, human autologous chondrocytes are used for implantation to treat cartilage defects. For chondrocyte culture, autologous serum is often used as supplement in the culture medium. It is under discussion, whether drawing the blood during anaesthesia for the cartilage biopsy harvest impairs the manufacturing of the autologous cell implant. Since many clinicians use the anaesthetic propofol, we investigated a possible impact of propofol on cell’s performance during cell expansion and 3D culture.

Methods and Materials

Blood was obtained from 10 donors (male/female 4/6, 30-49 years) before and after propofol administration (1% in soy oil, 2,3-4,2 mg/kg/h, 4-21 minutes after administration) to generate serum without and with propofol. Human primary chondrocytes were isolated from 3 donors (male/female 2/1, 56-76 years). Cell expansion and 3D spheroid formation were performed using both normal serum and serum with propofol (n=10). Cell adhesion was evaluated 5 days after seeding and gene expression of cartilage-matrix proteoglycan aggrecan (ACAN) was measured in chondrocyte spheroids after two weeks of culture.

Results

Serum with propofol was cloudy in 8/10 batches. Chondrocytes were expandable and 3D spheroids were formed using both serum with and without propofol. However, the propofol group showed lower cell adhesion in 5/10 batches (Figure 1A) and 3 of these showed less population doublings in P0 (Figure 1B) and thus additional passaging was necessary to achieve similar cell yields. ACAN gene expression was decreased in 3 spheroid cultures in the propofol group (Figure 2).

Conclusion

In presence of propofol, chondrocytes could still grow in 2D culture and assembled as spheroids in 3D culture. However, serum enriched with propofol may affect initial cell adhesion, to extend cell culture time, or change cartilage-specific gene expression in some chondrocyte cultures. Therefore, it is not recommended to draw the blood for autologous serum applications from the patient under anaesthesia with propofol.