title: Comparison of Matrigel and gelatin substrata for feeder-free culture of undifferentiated mouse embryonic stem cells for toxicity testing.
abstract: 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.
authors.affiliation: Marshfield Clinic Research Foundation, 1000 North Oak Avenue, Marshfield, WI 54449, USA. greenlee@ohsu.edu
authors.forename: A R
authors.initials: AR
authors.lastname: Greenlee
authors.forename: T A
authors.initials: TA
authors.lastname: Kronenwetter-Koepel
authors.forename: S J
authors.initials: SJ
authors.lastname: Kaiser
authors.forename: K
authors.initials: K
authors.lastname: Liu
mesh_terms: 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*
chemical_list: 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