Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.
BACKGROUND
Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC.


METHODS
PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation.


RESULTS
PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic medium was irrelevant as compared to PL and PI-PL.


CONCLUSIONS
The replacement of animal additives with human supplements is a basic issue in MSC ex vivo production. PI-PL represents a standardized, plasma-poor, human preparation which appears as a safe and good candidate to stimulate MSC growth in clinical-scale cultures.
Paola
P
Iudicone
Daniela
D
Fioravanti
Giuseppina
G
Bonanno
Michelina
M
Miceli
Claudio
C
Lavorino
Pierangela
P
Totta
Luigi
L
Frati
Marianna
M
Nuti
Immunohematology and Transfusion Medicine, San Camillo Forlanini Hospital, Rome, Italy. lpierelli@me.com.
Luca
L
Pierelli
D000818:Animals; D000941:Antigens / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D002457:Cell Extracts / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007165:Immunosuppression Therapy; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects; D010949:Plasma / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000187:drug effects / Q000276:immunology
D000941:Antigens; D002457:Cell Extracts; D036341:Intercellular Signaling Peptides and Proteins