Purpose

Development of novel allogeneic chondrocyte therapies are needed to provide a more widespread, cost-effective cartilage treatment option. Here we investigate the potential of juvenile cartilage sources for allogeneic chondrocyte manufacture.

Methods and Materials

Juvenile polydactyly digit (PD; n=4; aged 1±1 years (med±IQR)) or iliac apophysis cartilage (IA; n=6; aged 1±0.5years (med±IQR)) was used to derive chondrocyte cultures. Juvenile chondrocyte growth was compared to adult chondrocytes used for Autologous Chondrocyte Implantation (n=11; aged 41±9 years (med±IQR)). Further, juvenile chondrocytes (PD, n=2; IA, n=3) were up-scale manufactured using the Quantum® hollow-fibre bioreactor and compared to traditional tissue culture plastic (TCP) methods. All data are mean±SD.

Results

Comparable chondrocyte yields were obtained from juvenile (IA: 4.3±3.8x10³ cells/mg tissue; polydactyly: 4.2±4.2x10³ cells/mg tissue) and adult (2.6±0.1x10³ cells/mg tissue; p>0.05; One-Way Anova) sources. In contrast, doubling time (DT) (passage 1-3) for PD chondrocytes grown on TCP (2.66±1.57days) was significantly lower than IA (5.17±2.66 days) and adult chondrocytes (9.98±10.29 days) (Paired t-tests; p<0.05). Up-scale bioreactor expansion yielded 74.5±30 x10⁶ PD and 76±14x10⁶ IA chondrocytes in 11±1 days. DT was longer in the bioreactor cf. TCP (IA: bioreactor DT= 3.9±0.2 days, TCP DT= 2.0±0.3 days, t-test, p<0.05; PD: bioreactor DT=3.8±1.0 days, TCP DT=1.3±0.0 days). Juvenile chondrocytes were immunopositive (>95%) for CD90, CD73, CD44, CD166 and CD151 and immunonegative (<2%) for CD19, CD34, and CD45 and no difference in immunoprofile was observed cf. TCP expansion (One-Way ANOVA).

Conclusion

Juvenile chondrocytes represent attractive allogeneic cells sources, yielding large numbers of chondrocytes. However, our preliminary analyses indicate that their growth may be slowed upon hollow-fibre bioreactor expansion. Further analysis of key chondrogenic genes and in vitro cartilage forming capacity needs to be conducted in more donors to determine whether chondrogenic potential is influenced by up-scale manufacture.

Purpose

*SB and CHH joint first authors

Autologous Chondrocyte Implantation (ACI) requires a cartilage harvest and a subsequent cell implantation. There is a need to move towards therapies which are less invasive for patients and more cost-effective for health service providers. Allogeneic cells are one potential solution, but in order to manufacture them, alternatives for the autologous serum currently used in ACI protocols must be sought. To future-proof this process, a move towards xeno-free and serum-free protocols is also needed.

Methods and Materials

Chondroprogenitors were isolated from full depth human knee cartilage using selective adhesion to fibronectin and cryopreserved at passage 2-3 prior to use in these experiments. One ‘healthy’ cadaveric donor (a 22yo with no history of osteoarthritis/injury) and n=3 arthroplasty donors (mean 72yo) were included. Two xeno-free and serum-free GMP compliant culture media were tested for chondroprogenitors expanded on vitronectin (StemMacs MSC Expansion (media A; Miltenyi Biotech) and StemPro MSC Expansion (media B; Gibco)) cf. standard culture in DMEM/F12 with foetal bovine serum (FBS). Growth kinetics, immunoprofiling, gene expression analysis (RT-PCR) (n=4 donors) and chondrogenic analyses (n=2 donors) were performed for chondroprogenitors grown over passages 3 and 4.

Results

Chondroprogenitor manufacture in xeno-free and serum-free media does not significantly impact on their growth kinetics (population doubling level), immunoprofile (CD90, 73, 105, 44, 49c, 151, 29, 19, 34, 45, 49b, 14, 49a, 39 and HLA-DR) and, crucially, chondrogenic potential (GAG/DNA content), compared to FBS supplemented cultures. When expressed relative to FBS cultures, SOX-9 was significantly higher in media A cf. media B at passage 3. No other differences were found between expression levels in media A cf. B for any of the other genes tested (COLII, ACAN, COLX and ALK-1).

Conclusion

Our preliminary data suggests that xeno-free and serum-free media can be used to manufacture allogeneic chondroprogenitors without negatively altering their growth or phenotype.