Comparison of Matrigel and gelatin substrata for feeder-free culture of undifferentiated mouse embryonic stem cells for toxicity testing.
Murine embryonic stem (mES) cells have been used to evaluate cytotoxicity and developmental injury following exposure to embryotoxic agents. However, maintaining a homogeneous population of undifferentiated mES cells for this purpose has been complicated by the need for continuous co-culture with murine embryonic fibroblast (mEF) cells or limited passaging on plastic surfaces coated with gelatin. Here, we compare the synthetic basement membrane Matrigel with 0.1% gelatin substratum for feeder-free propagation of undifferentiated mES cells. Biomarkers of pluripotentiality, chromosome number, caspase-3 expression, and cardiomyocyte differentiation were monitored for mES cells cultured on Matrigel or 0.1% gelatin up to passage 7 (P7). Our results suggest that choice of substratum had no significant effect on population doubling time, cell viability, stage-specific embryonic antigen-1 (SSEA-1) expression, or early passage formation of beating cardiomyocytes (all P>or=0.09). In other comparisons, however, Matrigel supported significantly higher synthesis of alkaline phosphatase (7.7x10(-3)+/-0.8 vs 6.6x10(-3)+/-0.8 units/liter/cell, respectively, P=0.012), overall expression of activated caspase-3 following exposure to 5, 10, 50, 100 and 500 parts per billion (ppb) sodium arsenite (P<0.0001), and percent development to beating cardiomyocytes at P7 (P=0.01). Together, our findings suggest that Matrigel shows promise as a substrate for feeder-free propagation of undifferentiated mES cells for embryotoxicity endpoints.
Greenlee
A R
AR
Marshfield Clinic Research Foundation, 1000 North Oak Avenue, Marshfield, WI 54449, USA. greenlee@ohsu.edu
Kronenwetter-Koepel
T A
TA
Kaiser
S J
SJ
Liu
K
K
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D018053:Arsenites / Q000633:toxicity; D001672:Biocompatible Materials; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D003094:Collagen; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004622:Embryo, Mammalian / Q000166:cytology; D005780:Gelatin; D007797:Laminin; D016256:Lewis X Antigen / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D011509:Proteoglycans; D017670:Sodium Compounds / Q000633:toxicity; D018675:Toxicity Tests / Q000379:methods*
D018053:Arsenites; D001672:Biocompatible Materials; D004338:Drug Combinations; D007797:Laminin; D016256:Lewis X Antigen; D011509:Proteoglycans; D017670:Sodium Compounds; C059485:matrigel; C017947:sodium arsenite; D005780:Gelatin; D003094:Collagen; D000469:Alkaline Phosphatase; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases