TY - JOUR
T1 - Methods for Expansion of Three-Dimensional Cultures of Human Embryonic Stem Cells Using a Thermoresponsive Polymer
AU - Chen, Xiaoli
AU - Harkness, Linda
AU - Jia, Zhongfan
AU - Prowse, Andrew
AU - Monteiro, Michael J.
AU - Gray, Peter P.
PY - 2018/3
Y1 - 2018/3
N2 - Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.
AB - Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.
KW - hPSC expansion
KW - human pluripotent stem cells
KW - suspension cultures
KW - thermoresponsive nanobridges
UR - http://www.scopus.com/inward/record.url?scp=85045519199&partnerID=8YFLogxK
U2 - 10.1089/ten.tec.2017.0331
DO - 10.1089/ten.tec.2017.0331
M3 - Article
C2 - 29239281
AN - SCOPUS:85045519199
VL - 24
SP - 146
EP - 157
JO - Tissue Engineering - Part C: Methods
JF - Tissue Engineering - Part C: Methods
SN - 1937-3384
IS - 3
ER -