Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.
BACKGROUND
Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).


METHODS
The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed.


RESULTS
The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties.


CONCLUSIONS
We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.
José Paulo
JP
Martins
Jorge Miguel
JM
Santos
Joana Marto
JM
de Almeida
Mariana Alves
MA
Filipe
Mariana Vargas Teixeira
MV
de Almeida
Sílvia Cristina Paiva
SC
Almeida
Ana
A
Água-Doce
Alexandre
A
Varela
Mari
M
Gilljam
Birgitta
B
Stellan
Susanne
S
Pohl
Kurt
K
Dittmar
Werner
W
Lindenmaier
Evren
E
Alici
Luís
L
Graça
Pedro Estilita
PE
Cruz
Helder Joaquim
HJ
Cruz
Rita Nogueira
RN
Bárcia
D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation / Q000009:adverse effects / Q000379:methods* / Q000592:standards; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D011786:Quality Control; D057905:Stem Cell Research; D020858:Tissue and Organ Harvesting / Q000009:adverse effects / Q000379:methods* / Q000592:standards; D014470:Umbilical Cord / Q000166:cytology*