Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions

Abstract

Background aims: Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL).

Methods: Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed.

Results: The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed.

Conclusions: We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness.

Keywords: Good Manufacturing Practice; human platelet lysate; inactivation; mesenchymal stromal cells; psoralen.

Figure 1

HPL and iHPL-MSCs showed differences in CFU-F and cellular morphology compared with FBS-MSCs. (A) CFU-F numbers of HPL (white triangle), iHPL (gray diamond) and FBS (black square)-MSCs plated at seeding densities of 10,000 cells/cm² and 100,000 cells/cm². Each symbol represents an experiment (n = 9), and no significant differences were observed. (B) Representative phase images at ×5 magnification of CFU-Fs in the three different conditions: HPL (a) and iHPL (b)-MSC CFU-Fs were denser, more homogeneous and populated with cells than were FBS-CFU-Fs (c). Moreover, HPL and iHPL-MSCs (d, e) reached confluence faster than did FBS (f)-MSCs. (C) Representative phase images at ×40 magnification show the MSCs in the three different culture conditions after 3 and 12 h from plating: HPL and iHPL-MSCs emitted prominent extensions (a, b) and formed spherical structures similar to embryoid bodies (d, e). FBS-MSCs show a polygonal morphology with large, jagged cytoplasm (c) and did not form three-dimensional structures (f).

Figure 2

HPL and iHPL-MSCs show higher proliferative potential than do FBS-MSCs. (A–C) Cumulative PD of HPL, iHPL and FBS-MSCs (dashes). Results are shown as the cPD value at the first three passages of each 16 independent experiments in the three culture conditions. Mean ± SD values are represented in each graph as a thick black line. HPL and iHPL-MSCs show a higher proliferative potential than do FBS-MSCs from the second passage of culture.

Figure 3

HPL and iHPL-MSCs show more embryonic stem cell marker proteins and messenger RNA expression than do FBS-MSCs. (Panel A for Oct-3/4 and Panel B for NANOG) On left side of the panel, immunocytochemistry analysis of embryonic markers in HPL (left column), iHPL (middle column) and FBS (right column)-MSCs (×40 magnification); on the right side of the panel, a representative experiment of RT-PCR: Oct-3/4 (a–c) and NANOG (g–i) were more expressed in HPL and iHPL-MSCs compared with FBS-MSCs in terms of both protein and messenger RNA. For immunocytochemistry, figures are representative of one of three experiments, and DAPI staining was used to evidence the nucleus of each analyzed cell (d–f, l–n). For RT-PCR, five experiments were conducted, but both panels show the most representative experiment.

Figure 4

MSC differentiation potential assay after 3 weeks of specific induction in MSCs in the three different conditions. Von Kossa staining (a–c) shows the presence of calcium oxalates in osteoblasts, oil red O shows intracytoplasmatic vacuoles in adipocytes (d–f) and alcian blue (g–i) shows the hyaluronic acid for chondrocytes, respectively, in HPL (left column), iHPL (middle column) and FBS (right column)-MSCs (n = 19).

Figure 5

Soft agar assay to exclude tumorigenesis potential in MSCs in the three different conditions (n = 3). HPL, iHPL and FBS-MSCs (a–c) did not form colonies, nor did whole BM used as a negative control (d–f) compared with primary osteosarcoma tumor cells used as a positive control (g–i).