Abstract
BackgroundIn most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC therapeutic efficacies are inconsistent, largely attributed to transplanted MSC difficulties both in engrafting at tissue sites and in retaining their therapeutic functions from suspension formulations. MSC functional components, including cell adhesion and cell–cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Cell sheet technology, exploiting temperature-responsive cell culture surfaces, permits cell harvest without cell protein damage. This study is focused on phenotypic traits of MSC sheets structurally and functionally to understand therapeutic benefits of cell sheets.Methods/resultsThis study verified cleaved cellular proteins (vinculin, fibronectin, laminin, integrin β-1, and connexin 43) and increased apoptotic cell death produced under standard trypsin harvesting treatment in a time-dependent manner. However, MSC sheets produced without trypsin using only temperature-controlled sheet harvest from culture plastic exhibited intact cellular structures. Also, MSCs harvested using enzymatic treatment (i.e., chemical disruption) showed higher pYAP expression compared to MSC sheets.ConclusionRetention of cellular structures such as ECM, cell–cell junctions, and cell–ECM junctions is correlated with human umbilical cord mesenchymal stem cell (hUC-MSC) survival after detachment from cell culture surfaces. Retaining these proteins intact in MSC cultures using cell sheet technology is proposed to enhance stem cell survival and their function in stem cell-based therapy.
Highlights
In most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function
Human umbilical cord-derived mesenchymal stem cell culture Banked human umbilical cord mesenchymal stem cell (hUC-mesenchymal stem cells (MSC)) isolated from the subepithelial layer of human umbilical cord tissue (Jadi Cell LLC, Miami, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMAX (Gibco), 1% MEM non-essential amino acids (NEAA) (Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco) [19]. hUC-MSC was incubated at 37 °C with 5% CO2 in a humidified chamber and passaged when cells reached confluence. hUC-MSCs passaged with TrypLE (Gibco) treatment for 5 min were sub-cultured in media at 3000 cells/cm2 between passages 4 and 6
Results hUC-MSC sheet preparation Cells cultured on Temperature-responsive cell culture dishes (TRCD) change morphology from rounded to spindle shape when attached to TRCD
Summary
In most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC functional components, including cell adhesion and cell–cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Contrary to supporting preclinical animal studies, clinical studies to date show minimal or only transient improvements in therapeutic effects [1]. Cells are harvested from these single-use plastic surfaces for therapeutic use [2]
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