Lauren S Richardson1, Poorna R Menon2, Ramkumar Menon3. 1. Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine & Perinatal Research, The University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX, 77555, USA; Department of Neuroscience, Cell Biology & Anatomy, The University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX, 77555, USA. Electronic address: lestaffo@utmb.edu. 2. Clear Falls High School, 4380 Village Way, League City, TX, 77573, USA. 3. Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine & Perinatal Research, The University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX, 77555, USA. Electronic address: ra2menon@utmb.edu.
Abstract
INTRODUCTION: To determine 3D growth of amnion membrane cells using soft substrate plates of various rigidities. METHODS: Amnion epithelial (AEC) and mesenchymal cells (AMC) were cultured on 6-well soft substrate plates coated with matrigel and elastomer with rigidities of 0.5, 2, 8, 16, and 64 kPa (n = 3 each). Controls were cells in standard culture conditions. Cell morphology, spheroids' and sheets' formations and viability (bright field microscopy and crystal violet staining), and cellular transitions (vimentin/cytokeratin-18 [CK-18] ratios) were analyzed. A Student t-test was used for statistical analyses. RESULTS: AECs in substrate rigidities between 2 and 8 kPa formed 3D features (spheroids and sheets) while retaining viability. Two kPa produced spheroids with epithelial characteristics (decrease in vimentin), and 8 kPa favored sheets. Transplantation and culture of AEC sheets with no matrix or elastomers, retained AECs' viability and maintained their epithelial characteristics. Optimum AMC growth was also between 2 and 8 kP A, with predominance of vimentin; however, AMCs did not form 3D structures. Lower and higher rigidities transitioned AMCs into AECs (decrease in vimentin). DISCUSSION: Matrix rigidities between 2 and 8 kPa produced 3D structures of AECs (spheroids and sheets), resembling amnion membranes' morphology and exhibiting regenerative capacity in utero. Although AMCs grew in similar rigidities, a lack of 3D structures support their dispersed character in the membrane matrix. Extreme rigidities transitioned AMCs into AECs, suggesting that AMCs are transient cells (reservoirs) in the matrix required for remodeling. Compromises in matrix rigidity can cause membrane dysfunction and lead to adverse pregnancy outcomes.
INTRODUCTION: To determine 3D growth of amnion membrane cells using soft substrate plates of various rigidities. METHODS: Amnion epithelial (AEC) and mesenchymal cells (AMC) were cultured on 6-well soft substrate plates coated with matrigel and elastomer with rigidities of 0.5, 2, 8, 16, and 64 kPa (n = 3 each). Controls were cells in standard culture conditions. Cell morphology, spheroids' and sheets' formations and viability (bright field microscopy and crystal violet staining), and cellular transitions (vimentin/cytokeratin-18 [CK-18] ratios) were analyzed. A Student t-test was used for statistical analyses. RESULTS:AECs in substrate rigidities between 2 and 8 kPa formed 3D features (spheroids and sheets) while retaining viability. Two kPa produced spheroids with epithelial characteristics (decrease in vimentin), and 8 kPa favored sheets. Transplantation and culture of AEC sheets with no matrix or elastomers, retained AECs' viability and maintained their epithelial characteristics. Optimum AMC growth was also between 2 and 8 kP A, with predominance of vimentin; however, AMCs did not form 3D structures. Lower and higher rigidities transitioned AMCs into AECs (decrease in vimentin). DISCUSSION: Matrix rigidities between 2 and 8 kPa produced 3D structures of AECs (spheroids and sheets), resembling amnion membranes' morphology and exhibiting regenerative capacity in utero. Although AMCs grew in similar rigidities, a lack of 3D structures support their dispersed character in the membrane matrix. Extreme rigidities transitioned AMCs into AECs, suggesting that AMCs are transient cells (reservoirs) in the matrix required for remodeling. Compromises in matrix rigidity can cause membrane dysfunction and lead to adverse pregnancy outcomes.
Authors: Lauren S Richardson; Gracie Vargas; Tyra Brown; Lorenzo Ochoa; Samantha Sheller-Miller; George R Saade; Robert N Taylor; Ramkumar Menon Journal: Am J Pathol Date: 2017-09-20 Impact factor: 4.307
Authors: N Athayde; R Romero; R Gomez; E Maymon; P Pacora; M Mazor; B H Yoon; S Fortunato; R Menon; F Ghezzi; S S Edwin Journal: J Matern Fetal Med Date: 1999 Sep-Oct
Authors: Ramkumar Menon; Istvan Boldogh; Hal K Hawkins; Michael Woodson; Jossimara Polettini; Tariq Ali Syed; Stephen J Fortunato; George R Saade; John Papaconstantinou; Robert N Taylor Journal: Am J Pathol Date: 2014-05-12 Impact factor: 4.307
Authors: Ramkumar Menon; Istvan Boldogh; Rheanna Urrabaz-Garza; Jossimara Polettini; Tariq Ali Syed; George R Saade; John Papaconstantinou; Robert N Taylor Journal: PLoS One Date: 2013-12-27 Impact factor: 3.240