2025 Volume 48 Issue 7 Pages 1107-1110
Mesenchymal stem cell (MSC) sheet therapies are effective in treating intractable diseases. Aligned and oriented bone-marrow-derived MSC sheets have been developed using stripe-patterned thermoresponsive cell culture dishes to increase the effectiveness of MSC therapies compared with unaligned MSC sheets. However, adipose-tissue- and umbilical-cord-derived MSCs (ADMSCs and UCMSCs, respectively) have not yet been used for preparing aligned cell sheets. Therefore, we prepared aligned cell sheets comprising ADMSCs and UCMSCs. We produced a patterned cell culture dish by modifying polyacrylamide into a striped pattern on a commercially available poly(N-isopropylacrylamide)-modified dish. Aligned cell sheets comprising ADMSCs and UCMSCs were successfully prepared in these culture dishes.
Mesenchymal stem cells (MSCs) have been studied for use as therapeutic cells.1–6) MSC therapies are effective in treating diverse symptoms because MSCs secrete various cytokines involved in cell proliferation, angiogenesis, inflammatory suppression, and immunoregulation.1–3) Most MSC transplantations are administered intravenously; therefore, the biodistribution of MSCs after transplantation cannot be controlled. MSCs do not remain effective at disease sites.
MSC sheets have been developed as cellular tissues to effectively increase the therapeutic efficiency of MSCs.7,8) MSC sheets can be prepared using poly(N-isopropylacrylamide) (PNIPAAm)-modified dishes. PNIPAAm exhibits temperature-responsive hydrophilic and hydrophobic changes attributed to hydration and dehydration at low and high temperatures, respectively. MSCs adhere to and proliferate on PNIPAAm-modified dishes at 37°C. MSC sheets can be obtained after the MSCs reach confluency by changing the temperature from 37 to 20°C, which causes the modified PNIPAAm on the dish to become hydrophilic and the MSC sheet to detach from the dish without requiring the use of digestive enzymes. The MSC sheet maintains its structure and high cell cytokine expression, which is attributable to the activity of the MSCs being retained by recovering them from the dish without digestive enzymes.9,10) Thus, the transplantation success rate is higher with MSC sheets than with MSC suspensions11) because the MSC sheets retain functional cell–cell junctions and an endogenous extracellular matrix.
Aligned cell sheets composed of bone-marrow-derived MSCs (BMMSCs) were prepared using a stripe-patterned thermoresponsive cell culture dish to strengthen the functioning of MSC sheets. The cytokine expression level in the prepared aligned BMMSC sheets was higher than that in the unaligned MSC sheets.12) However, only BMMSCs were considered in prior studies on preparing aligned MSC sheets.
The MSCs from other tissues, such as adipose-derived MSCs (ADMSCs) and umbilical-cord-derived MSCs (UCMSCs), have been studied for use as therapeutic agents. The cytokine expression behavior, adhesion, and proliferation properties differ between ADMSCs and UCMSCs.13) Thus, aligned ADMSC sheets and UCMSC sheets prepared using a patterned dish could be effectively applied as therapy.
In this study, we examined whether ADMSCs and UCMSCs could be produced in patterned temperature-responsive culture dishes (Fig. 1). Patterned temperature-responsive culture dishes were prepared through photopolymerizing acrylamide (AAm) in striped patterns on a PNIPAAm cell culture dish (Fig. 1A). An AAm solution containing a photoinitiator was added to a commercially available PNIPAAm dish. Visible light was irradiated through the striped pattern of the photomask (Supplementary Fig. S1). Polyacrylamide (PAAm) was grafted onto the light-irradiated area because AAm was polymerized using a photoinitiator (Supplementary Fig. S2).
The cells that did not adhere to the PAAm were relatively weak because PAAm is hydrophilic. In contrast, the cells tended to adhere to the PNIPAAm-exposed areas because PNIPAAm is hydrophobic. The cells initially adhered to the PNIPAAm-modified area and extended in a striped pattern after seeding. The cells proliferated along the PNIPAAm line. The cells occupied the PNIPAAm area and extended to the PAAm-modified area while maintaining their orientation, forming an oriented cell sheet (Fig. 1B). The orientation of the prepared ADMSC and UCMSC sheets was investigated using a patterned dish.
The fluorescent protein adsorption capacities of the prepared patterned cell culture dishes were evaluated (Fig. 2). The adsorption of fluorescein-labeled fibronectin and bovine serum albumin (BSA) onto the patterned dish was quantified to determine the line pattern on the prepared dish (Fig. 2). Overlay photographs of these 2 protein adsorption images were prepared to determine the protein adsorption location (Supplementary Fig. S3). Fibronectin and bovine serum albumin were selectively adsorbed onto the striped PNIPAAm-exposed areas in the dish. The adsorption of proteins was uniformly distributed on the dish without the PAAm coating. The PAAm-modified area in the dish was relatively hydrophilic compared with the unmodified area, forming a striped pattern of alternating protein-adsorption and low-protein-adsorption areas. The BSA staining was broader than the fibronectin staining, which was probably due to the weak adsorption properties of BSA on PNIPAAm compared with those of fibronectin. Thus, BSA diffused into the solution, leading to a broader stained area.
Scale Bar: 100 μm.
A stripe-patterned PAAm layer was successfully prepared on a dish via photopolymerization.
Orientation of Aligned MSC Sheet Derived from Adipose and Umbilical Cord TissueADMSC and UCMSC sheets were prepared using patterned cell culture dishes. A phase-contrast image of an MSC sheet is shown in Fig. 3A. ADMSC and UCMSC sheets were prepared using nonpatterned cell culture dishes for comparison (Supplementary Fig. S4A). The MSCs were confluent, extended in one direction, and formed cell sheets owing to the striped pattern of the culture dish, whereas the MSCs were not oriented on the nonpatterned dishes. The MSCs could not be observed during initial adhesion and proliferation because they tended to detach from the patterned dish while capturing the microscopy images. In a previous study on a similarly patterned dish, the normal human dermal fibroblasts (NHDFs) adhered to the PNIPAAm-modified region, elongated, and then proliferated in a striped pattern, occupying the PNIPAAm region while maintaining their orientation.14) The same occurred in our study. The MSCs occupied the PNIPAAm area and extended to the PAAm-modified area while maintaining their orientation, forming an oriented cell sheet on a patterned dish. The results indicated that the patterned dish could be used to prepare an oriented cell sheet composed of MSCs derived from adipose and umbilical cord tissues.
A histogram of the cell orientation in the MSC sheet was obtained from the phase-contrast image of the cell sheet (Fig. 3B). ADMSC and UCMSC sheets were prepared using nonpatterned cell culture dishes for comparison (Supplementary Fig. S4B). The histograms of MSCs on the patterned dishes exhibited sharp peaks centered at 0°. Conversely, the MSCs cultured on nonpatterned dishes exhibited no peaks in the histogram. Most of the MSCs adhered to the culture dish and extended in one direction on the patterned dish. The ADMSCs exhibited a sharper peak in the histograms than the UCMSCs, which was probably due to the size difference between the ADMSCs and UCMSCs 19.715) and 15.8 μm, respectively. ADMSCs have a large surface area for adhering to cell culture dishes and are more likely to adopt an oriented state during extension and growth.
These results indicate that the patterned thermoresponsive cell culture dish could be used to prepare oriented cell sheets composed of ADMSCs and UCMSCs in MSC sheet therapy. The cytokine expression levels of the oriented ADMSC and UCMSC sheets will be investigated in future studies to determine the diseases that can be effectively targeted with these MSC sheets.
Oriented cell sheets comprising ADMSCs and UCMSCs were prepared in stripe-patterned thermoresponsive cell culture dishes via PAAm modification of a PNIPAAm-modified dish. The fluorescent protein adsorption onto the prepared dish indicated that cell-adhesive and weakly cell-adhesive areas were present in the dish. ADMSCs and UCMSCs were seeded in a patterned dish and cultured until confluence. These results indicate that patterned thermoresponsive cell culture dishes can be used to prepare oriented ADMSC and UCMSC sheets for targeted therapies.
Patterned temperature-responsive culture dishes were prepared via AAm with lined patterns on a PNIPAAm cell culture dish, as previously described12) (Fig. 1A). All reagents are described in Supplementary Materials S1. The detailed procedure for preparing the patterned dish is described in Supplementary Materials S3. In brief, AAm solution containing a photoinitiator was added to a commercially available PNIPAAm dish. Then, the dishes were irradiated with visible light through a stripe-patterned photomask (Supplementary Fig. S1), leading to the selective PAAm modification of PNIPAAm. The prepared dishes were washed with pure water. The striped pattern was observed using fluorescently labeled fibronectin and BSA.
Preparing MSC SheetsThe detailed cell culture and cell sheet preparation methods are described in Supplementary Materials S2 and S4. Briefly, 2 mL of ADMSC or UCMSC suspension (4.0–4.5 × 104 cells/mL) was seeded into the patterned dish. The dish was incubated at 37°C for 3–4 d until the MSCs proliferated and reached confluence. The MSC sheets were observed using a phase-contrast microscope. The degree of orientation of the MSCs in the sheet in the phase-contrast image was analyzed using the ImageJ software (NIH, Bethesda, MA, U.S.A.).
This study was supported by Grants-in-Aid for Scientific Research (Grant Numbers: JP21KK0199, JP22K19899, and JP24K01181) from the Japan Society for the Promotion of Science, the Precise Measurement Technology Promotion Foundation (PMTP-F), the CASIO Science Promotion Foundation, and the Mukai Science and Technology Foundation.
The authors declare no conflict of interest.
This article contains supplementary materials.
