RESUMEN
Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins, human and animal sera matrices, and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media, human foreskin fibroblast-conditioned culture medium, chemically defined medium, TeSR1, and modified TeSR1 media. The results showed the undefined, xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins, serum matrices, and the biomaterials tested. A long-term, feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium, but a xeno-free, fully defined, and reproducible feeder cell-free hESC culture method still remains to be developed.
Asunto(s)
Materiales Biocompatibles/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Forma de la Célula/efectos de los fármacos , Sistema Libre de Células , Colágeno/farmacología , Ensayo de Unidades Formadoras de Colonias , Combinación de Medicamentos , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Laminina/farmacología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteoglicanos/farmacología , SueroRESUMEN
BACKGROUND: Human embryonic stem cells (hESC) are excellent candidates for cell replacement therapies. However, currently used culture conditions contain animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hESC. METHODS: Nine xeno-free culture media were compared with the conventional serum replacement (ko-SR) containing media in the culture of hESC on human feeder cells. Cultured hESC were characterized immunocytochemically and by fluorescence-activated cell sorter analysis. The differentiation potential of hESC cultured with xeno-free media was determined with the RT-PCR analysis. RESULTS: The hESC cultured in xeno-free media differentiated or the proliferation decreased substantially. Under some test conditions, the morphology of the feeder cells was altered considerably. The hESC cultured with human serum underwent excessive differentiation in the beginning of culture, but a fraction of hESC was able to adapt to culture conditions containing 20% of human serum. CONCLUSIONS: None of the studied xeno-free media was able to maintain the undifferentiated growth of hESC. The medium containing 20% human serum was found to sustain undifferentiated hESC proliferation to some extent, yet was inferior to the conventional ko-SR-containing medium.