420: Routine expansion of mesenchymal stem cells derived from pooled rat amniotic fluid

Abstract

ObjectiveEvaluation of rat amniotic fluid (AF) as a source for routine expansion of mesenchymal stem cell (AF-MSC). Culture conditions have an important influence on expansion of AF-MSC as well as their later properties. Given poorer results of lower density cell protocol, we will also evaluated within this study, the feasibility of rat AF-MSC expansion using a high cell density protocol (HCDP) with two different culture media.Study DesignWe used AF from 10 E19 pregnant Wistar rats. Following midline laparotomy and hysterotomy, the sacs were punctured. AF from all amniotic sacs from the same dam was pooled to obtain enough amniotic cells. Cells were seeded using a density of 105cells/cm2. Culture medium was containing either fetal bovine serum (FBS; Invitrogen, Merelbeke, Belgium) as used for expansion of human AF-MSC (Ochsenbein-Kolble et al, 2003), or FBS optimized for human MSC culture (Stem Cell Technologies, Grenoble, France). Primary outcome measure was self-renewal ability. AF-MSC were characterized after >20 cell doublings by morphology, fluorescence activated cell scanning and differentiation assays.ResultsCell growth was observed in 100% of the AF samples without difference between the two culture media. Mesenchymal surface markers (CD90, CD44, CD31, CD29) were positive. Osteo- and adipo-differentiation were both demonstrated using immunohistochemical stainings (respectively Alizarine Red S and Oil Red staining).ConclusionsWe demonstrated that AF-MSC can be easily isolated and expanded from rat AF, without any difference between the used culture media. HCDP is an efficient and cheaper alternative to FBS optimized for human MSC culture without difference in later cell properties. Work supported by a grant of the European Commission (6th Framework Programme www.eurostec.eu, LSHC-CT-2006-037409 ). ObjectiveEvaluation of rat amniotic fluid (AF) as a source for routine expansion of mesenchymal stem cell (AF-MSC). Culture conditions have an important influence on expansion of AF-MSC as well as their later properties. Given poorer results of lower density cell protocol, we will also evaluated within this study, the feasibility of rat AF-MSC expansion using a high cell density protocol (HCDP) with two different culture media. Evaluation of rat amniotic fluid (AF) as a source for routine expansion of mesenchymal stem cell (AF-MSC). Culture conditions have an important influence on expansion of AF-MSC as well as their later properties. Given poorer results of lower density cell protocol, we will also evaluated within this study, the feasibility of rat AF-MSC expansion using a high cell density protocol (HCDP) with two different culture media. Study DesignWe used AF from 10 E19 pregnant Wistar rats. Following midline laparotomy and hysterotomy, the sacs were punctured. AF from all amniotic sacs from the same dam was pooled to obtain enough amniotic cells. Cells were seeded using a density of 105cells/cm2. Culture medium was containing either fetal bovine serum (FBS; Invitrogen, Merelbeke, Belgium) as used for expansion of human AF-MSC (Ochsenbein-Kolble et al, 2003), or FBS optimized for human MSC culture (Stem Cell Technologies, Grenoble, France). Primary outcome measure was self-renewal ability. AF-MSC were characterized after >20 cell doublings by morphology, fluorescence activated cell scanning and differentiation assays. We used AF from 10 E19 pregnant Wistar rats. Following midline laparotomy and hysterotomy, the sacs were punctured. AF from all amniotic sacs from the same dam was pooled to obtain enough amniotic cells. Cells were seeded using a density of 105cells/cm2. Culture medium was containing either fetal bovine serum (FBS; Invitrogen, Merelbeke, Belgium) as used for expansion of human AF-MSC (Ochsenbein-Kolble et al, 2003), or FBS optimized for human MSC culture (Stem Cell Technologies, Grenoble, France). Primary outcome measure was self-renewal ability. AF-MSC were characterized after >20 cell doublings by morphology, fluorescence activated cell scanning and differentiation assays. ResultsCell growth was observed in 100% of the AF samples without difference between the two culture media. Mesenchymal surface markers (CD90, CD44, CD31, CD29) were positive. Osteo- and adipo-differentiation were both demonstrated using immunohistochemical stainings (respectively Alizarine Red S and Oil Red staining). Cell growth was observed in 100% of the AF samples without difference between the two culture media. Mesenchymal surface markers (CD90, CD44, CD31, CD29) were positive. Osteo- and adipo-differentiation were both demonstrated using immunohistochemical stainings (respectively Alizarine Red S and Oil Red staining). ConclusionsWe demonstrated that AF-MSC can be easily isolated and expanded from rat AF, without any difference between the used culture media. HCDP is an efficient and cheaper alternative to FBS optimized for human MSC culture without difference in later cell properties. Work supported by a grant of the European Commission (6th Framework Programme www.eurostec.eu, LSHC-CT-2006-037409 ). We demonstrated that AF-MSC can be easily isolated and expanded from rat AF, without any difference between the used culture media. HCDP is an efficient and cheaper alternative to FBS optimized for human MSC culture without difference in later cell properties. Work supported by a grant of the European Commission (6th Framework Programme www.eurostec.eu, LSHC-CT-2006-037409 ).