Comparative analyses of industrial-scale human platelet lysate preparations

Abstract

Background: Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product.

Study design and methods: Nine industrial-scale, serum-based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet-rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma-based or serum-based PL were compared to MSCs cultured with fetal bovine serum.

Results: Electrolyte and protein levels were relatively consistent among all serum-based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum-based PL stored at -80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum-based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement.

Conclusion: Using a large-scale, standardized method, lot-to-lot variations were noted for industrial-scale preparations of serum-based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off-the-shelf PL is a feasible substitute for fetal bovine serum in MSC cultures.

Figure 1. Biochemical and growth factor compositions of industrial scale platelet lysate lots

Aliquots from PL-S lots (average 18.8±2.5 liters) were tested for A) sodium, chloride, urea nitrogen, glucose, calcium, and osmolality; B) potassium, creatinine, pH, total protein and hemoglobin levels. (n=9); C) platelet derived growth factor isoform (PDGF-BB), epidermal growth factor (EGF) D) vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The concentration of each growth factor from 6 of 9 lots was determined by an enzyme-linked immunosorbent assay (ELISA).

Figure 2. PL-S/FBS growth ratios for MSCs

MSCs were cultured with 10% PL-S or 10% FBS. MSC proliferative responses were measured by either a manual cell count (black bars) or by using an MTT assay (white bars). Proliferative responses of MSCs when cultured with PL-S lots 1, 2, 3, 4, 5, & 7 were determined at passage 3 by performing manual cell counts of MSCs after staining the cells with trypan blue. Growth ratios were then calculated for these lots by dividing the total viable MSC counts from cultures supplemented with 10% PL-S by the total viable MSC counts from cultures supplemented with FBS (black bars). Proliferative responses during passage 3 MSCs formation forots 7, 8 &9 were determined by using an MTT assay Growth ratios were calculated by dividing the average absorbance of wells containing MSCs cultured with PL-S by the average absorbance of wells containing MSCs cultured with FBS (white bars). ND=not done

Figure 3. PL-S stability testing over a 2-year period

Three randomly selected industrial scale lots were selected for stability testing. A) fold-expansions, and B) doubling-times were determined from each of the 3 lots of PL-S that were tested immediately after the completion of the PL-S manufacturing run (i.e. time zero) or after 1–10 mL aliquots from each PL-S lot were maintained at −80°C for 6, 12 or 24 months. A single lot of FBS was used and tested over the same time period. Using a single lot of MSC for all time points, MSCs that had been stored frozen were thawed and plated in 6 well plates that contained basal medium supplemented with either PL-S or with FBS. After cells reached confluence, they were harvested to determine fold-expansions and doubling times. C) The concentration of PDGF-BB, VEGF, EGF and bFGF in aliquots of PL-S were assessed upon completion of the PL-S manufacturing run or after being maintained at −80°C for 6, 12 or 24 months. (n=3)

Figure 4. Hierarchical heat map clusteri analysis showing individual protein expression levels in PL-S lots

Cluster analysis was performed for 3 different lots of PL-S. Low expression protein values are represented as blue, high expression protein values are designated as yellow, and intermediate values are black. Dendrogram shows the similarity of the groups.

Figure 5. Transcriptome analysis of MSCs

A) MSCs were prepared from the marrow of two living donors and one cadaveric donor. The marrow from one living donor was directly plated into culture medium. The Marrow from a second living donor was processed to obtain an MNC fraction, which was then plated. And, the marrow from an organ donor was processed to obtain a MNC fraction that was stored frozen in small aliquots prior to being plated. Each source of marrow was cultured with MEM medium supplemented with 10% FBS, 10% PL-P, or 10% PL-S. Principal component analysis (PCA) of the samples using the entire gene expression data set is shown in on the left and unsupervised hierarchical clustering analysis is shown on the right. B) Transcriptome analysis of MSCs prepared from the marrow of 4 different living donors. Transcriptome analysis of MSCs produced in media supplemented with 10% FBS and PL-S. Marrow aspirates from 4 different healthy subjects were process to obtain a MNC fraction. One-half of the MNC fraction was plated in αMEM medium supplemented 10% FBS and the other half with 10% PL-S. Principal component analysis (PCA) of the samples using the entire gene expression data set is shown on the left and unsupervised hierarchical clustering analysis is shown on the right. MSCs cultured in 10% FBS are shown in red and those cultured in 10% PL-S are shown in blue. D1 denotes donor 1, D2 donor 2, D3 donor 3 and D4 donor 4.