Human Platelet Lysate as an Alternative to Autologous Serum for Human Chondrocyte Clinical Use

Abstract

Objective: A pivotal aspect of cartilage tissue engineering resides in cell culture medium supplementation, in view of maximizing in vitro cell proliferation and preserving cellular functionality. Autologous human serum (aHS) is commonly used as an inducive supplement for safe human articular chondrocyte (HAC) proliferation prior to clinical implantation. However, practical clinical use of aHS is hindered by constraining manufacturing requirements and quality assurance-driven downstream processing. The present study investigated potential alternative use of commercial human platelet lysate (hPL) supplements in HAC manufacturing workflows related to clinical therapeutic pathways.

Design: Differential effects of hPL, aHS, and fetal bovine serum were assessed on primary cultured HAC parameters (viability, proliferative rates, and morphology) in 2-dimensional in vitro systems. A 3-dimensional HAC pellet model served for postexpansion assessment of cellular functionality, by visualizing proteoglycan production (Alcian blue staining), and by using qRT-PCR relative quantification of chondrogenic marker (SOX9, COL2-A1, and ACAN) genetic expression.

Results: We found that monolayer HAC culture with hPL or aHS supplements presented similar characteristics (elongated cell morphology and nearly identical growth kinetics). Chondrogenic activity appeared as conserved in HACs expanded with human or bovine supplements, wherein histologic analysis indicated a progressive sGAG accumulation and SOX9, COL2-A1, ACAN gene expression was upregulated in 3-dimensional HAC pellet models.

Conclusion: This study therefore supports the use of hPL as a functional equivalent and alternative to aHS for cultured HAC batch preparation, with the potential to effectively alleviate pressure on clinical and manufacturing bottlenecks in cell therapy approaches for cartilage regeneration.

Keywords: autologous chondrocyte culture; cartilage repair; cell therapy; chondrocyte transplantation; human platelet lysate.

Figure 1.

Morphology of HACs expanded in monolayer culture. HACs were isolated from articular cartilage samples and cultured in F12:DMEM supplemented with 10% FBS, 10% hPL, or 10% aHS. Scale bars represent 0.2 mm. HAC, human articular chondrocyte; F12:DMEM, Ham’s F12–Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; hPL, human platelet lysate; aHS, autologous human serum.

Figure 2.

Chondrogenesis assessment in 3-dimensional pellets. Chondrogenic differentiation was histologically assessed at day 7 and day 14 of induction. Three-dimensional HAC pellets were stained with Alcian blue and counter-stained with Nuclear Fast Red. Scale bars represent 0.02 mm. HAC, human articular chondrocyte.

Figure 3.

(A) Short-term HAC proliferation. HACs were cultured in the presence of hPL, aHS, or FBS. Data were presented as average ± SD, n = 10, paired experiments, *P < 0.05, ***P < 0.001, NS nonsignificant. (B) Long-term HAC proliferation. HACs were cultured in the presence of hPL. Data present cell counts and population doubling times (PDT) at 85% confluency. Data are presented as average ± SD, n = 16, independent experiments. (C) Chondrogenic marker gene expression measured by qRT-PCR in HAC pellets. Data are presented using induction folds, wherein box plots indicated medians and quartiles, and the whiskers represented minima and maxima, *P < 0.05. HAC, human articular chondrocyte; FBS, fetal bovine serum; hPL, human platelet lysate; aHS, autologous human serum.