title: Feeder cell density--a key parameter in human embryonic stem cell culture.
abstract: A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm2, above the recommended value of 20,000 cells/cm2, appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm2), the ES cells appear to stack up to form a "bulge." This was not observed under the recommended feeder cell density of 20,000 cells/cm2. By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm2. This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.
authors.affiliation: Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, 119074 Singapore.
authors.forename: Boon Chin
authors.initials: BC
authors.lastname: Heng
authors.forename: Hua
authors.initials: H
authors.lastname: Liu
authors.forename: Tong
authors.initials: T
authors.lastname: Cao
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
references.citation: Stem Cells. 2003;21(2):131-42
references.pmid: 12634409
references.citation: Stem Cells. 2003;21(5):546-56
references.pmid: 12968109
references.citation: Stem Cells. 2004;22(1):51-64
references.pmid: 14688391
references.citation: Science. 1998 Nov 6;282(5391):1145-7
references.pmid: 9804556
references.citation: Nature. 1981 Jul 9;292(5819):154-6
references.pmid: 7242681
references.citation: Nat Biotechnol. 2000 Apr;18(4):399-404
references.pmid: 10748519
references.citation: Biol Reprod. 2003 Jun;68(6):2150-6
references.pmid: 12606388
references.citation: N Engl J Med. 2004 Mar 25;350(13):1353-6
references.pmid: 14999088
references.citation: Transpl Immunol. 2004 Apr;12(3-4):203-9
references.pmid: 15157914
references.citation: Hum Reprod. 2004 Mar;19(3):676-84
references.pmid: 14998970
references.citation: Nat Biotechnol. 2004 Jun;22(6):707-16
references.pmid: 15146197
references.citation: Nat Biotechnol. 2002 Sep;20(9):933-6
references.pmid: 12161760
references.citation: Biol Reprod. 2004 Mar;70(3):837-45
references.pmid: 14627547
references.citation: Hum Reprod. 2003 Jul;18(7):1404-9
references.pmid: 12832363
references.citation: Biol Reprod. 2004 Dec;71(6):1772-8
references.pmid: 15140800
references.citation: Biol Reprod. 2003 Dec;69(6):2007-14
references.pmid: 12930726
references.citation: Nat Biotechnol. 2001 Oct;19(10):971-4
references.pmid: 11581665
references.citation: Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8
references.pmid: 6950406