Comparison of Human Platelet Lysate versus Fetal Bovine Serum for Expansion of Human Articular Cartilage-Derived Chondroprogenitors

Abstract

Purpose: Articular chondroprogenitors, a suitable contender for cell-based therapy in cartilage repair, routinely employ fetal bovine serum (FBS) for expansion and differentiation. The possibility of transplant rejections or zoonoses transmissions raise a need for xeno-free alternatives. Use of human platelet lysate (hPL), a nutrient supplement abundant in growth factors, has not been reported for human chondroprogenitor expansion thus far. Our aim was to compare the biological profile of chondroprogenitors grown in hPL versus FBS.

Methods: Chondroprogenitors were isolated from 3 osteoarthritic knee joints. Following differential fibronectin adhesion assay, passage 0 cells grown in (a) 10% FBS and (b) 10% hPL were considered for assessment of growth kinetics, surface marker expression, gene expression, and trilineage differentiation. Latent transforming growth factor-β1 (TGFβ1) levels were also measured for each culture medium used.

Results: Cellular proliferation was significantly higher in cells grown with hPL (P < 0.01). Surface marker expression was comparable except in CD-146 where hPL group had significantly higher values (P = 0.03). Comparison of mRNA expression revealed notably low values of collagen I, collagen X, aggrecan, and collagen II (P < 0.05). Trilineage differentiation was seen in both groups with higher alizarin red uptake noted in hPL. There were also significantly higher levels of latent TGFβ1 in the medium containing hPL as compared to FBS.

Conclusions: This is the first in vitro xeno-free study to affirm that hPL can serve as an optimal growth supplement for expansion of articular chondroprogenitors, although an in-depth assessment of resident growth factors and evaluation of different dilutions of hPL is required to assess suitability for use in translational research.

Keywords: chondroprogenitors; fetal bovine serum; human platelet lysate.

Figure 1.

Algorithm depicting sequence of cellular isolation, culture condition using either FBS or HPL and its subsequent evaluation parameters. FBS, fetal bovine serum; hPL, human platelet lysate.

Figure 2.

Representative clonally derived human articular CPs following FAA cultured with FBS/hPL. (A, E): A clone at day 3 forming a cluster of 2 to 3 cells (10×). (B, F): Clone cluster at day 7 (20×). (C) Clonal growth >5 population doubling. (G): Nonclonal growth at day 11 (10×). (D, H): Passage 0 cultures with honeycomb growth pattern observed with CPs grown in hPL (10×). CP, chondroprogenitor; FAA, fibronectin adhesion assay; FBS, fetal bovine serum; hPL, human platelet lysate.

Figure 3.

Data representing cumulative population doubling (CPD) values for 2 subgroups (n = 3 human samples) across time in culture (p0-p3). Data expressed as mean ± SD. FBS, fetal bovine serum; hPL, human platelet lysate. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4.

CP cell cycle analysis with DAPI using Watson model. (A, B). Representative FACS plots of phase distribution. (C, D) Pie chart depicting phase distribution of mean scores (n = 3 human samples). CO, chondroprogenitor; FBS, fetal bovine serum; hPL, human platelet lysate.

Figure 5.

β-Galactosidase assay comparing the number of presenescent and senescent cells between the chondroprogenitors grown with FBS and hPL. Positive uptake indicated light blue stain. Magnification: 10×. FBS, fetal bovine serum; hPL, human platelet lysate.

Figure 6.

Comparison of percentage expression of CD markers between FBS and hPL at passage 1 (n = 3 human samples). Data expressed as mean ± SD. *P < 0.05. FBS, fetal bovine serum; hPL, human platelet lysate.

Figure 7.

Relative expression of SOX9, collagen type II, aggrecan, collagen type I, RUNX2, MMP-13, and collagen type X between FBS and hPL groups. ΔCt values normalized to GAPDH are expressed as mean ± SD (*P < 0.05, **P < 0.01). FBS, fetal bovine serum; hPL, human platelet lysate.

Figure 8.

Trilineage differentiation of chondroprogenitors from osteoarthritic joints between FBS and hPL groups. Representative microscopic images of oil red O (A-B, G-H) and Alizarin red (C-D, I-J) staining to confirm adipogenic and osteogenic differentiation (10×). Chondrogenic differentiation was confirmed by Alcian blue (E, K) and collagen II (F, L) staining of formed cell pellets (10×).