Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets

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.

Trends in Articular Cartilage Tissue Engineering: 3D Mesenchymal Stem Cell Sheets as Candidates for Engineered Hyaline-Like Cartilage

“…Although in vitro chondrogenic differentiation is extensively published for pellet cultures, cell seeded scaffolds, and scaffold-free high-density seeding cultures [ 14 , 15 , 16 , 87 ], these 3D constructs are limited in their abilities to achieve both robust hyaline-like differentiation and direct, unassisted transplantation to defect sites [ 1 , 21 , 23 , 154 ]. To address these concerns, cell sheet tissue engineering constructs afford improved control of 3D cellular interactions, maintenance of chondrogenic characteristics via established manipulation techniques, and optimize endogenous adhesion abilities [ 83 , 156 , 157 , 160 , 162 , 163 , 164 ]. To date, autologous chondrocyte cell sheets have exhibited experimental and some clinical success in adhering, surviving, and inducing regeneration in articular cartilage defects [ 24 , 173 , 177 , 180 , 182 , 185 , 189 ].…”

“…Below the PIPAAm lower critical solution temperature (32 °C), cells spontaneously detach from the culture surface, bypassing typical culture requirements for damaging enzymatic cell harvesting [ 160 , 162 ]. This temperature-mediated detachment retains endogenous cell–cell and cell-ECM interactions and preserves cellular environments, allowing cultured cells to be harvested as intact cell sheets [ 83 , 156 , 157 , 160 , 162 , 163 , 164 ]. As cells are seeded and grown under adherent 2D conditions, this abrupt temperature-mediated detachment prompts established cytoskeletal filaments and retained ECM to naturally contract when released from culture surfaces [ 165 , 166 ].…”

Adhesion of Epithelial Cells to PNIPAm Treated Surfaces for Temperature-Controlled Cell-Sheet Harvesting

“… 67 This also highlights the benefit of PMS usage, as vinculin was shown to be preserved when using PNIPAm for subculture over mechanical or chemical dissolution. 8 Thus after 48 h and in contrast to the early adhesion phase, cell–cell contact forces based on cadherins should dominate this culture phase.…”

“… 5 − 7 Often cell adhesion on standard cell culture surfaces is so strong that cell detachment requires a chemical or physical treatment with potentially harmful enzymes or mechanical force. 8 A more gentle approach is controllable cell adhesion on designed substrates in the more unique way of cell-sheet engineering. It fabricates a monolayer of cells and has been described to maintain extracellular structures with minimal damage to the cell morphology and function for tissue reconstruction.…”

Novel therapies using cell sheets engineered from allogeneic mesenchymal stem/stromal cells

“…Cell sheets engineered from human MSCs (i.e. MSC sheets) have been studied in vitro [ 101 , 102 ] and in vivo to employ those functional properties. Significantly, cell sheet technology enables transplantation of tissue-like structures containing contiguous MSCs grown in their endogenous matrix onto target sites without off-target distribution.…”