Poster K18 - Expanding Research in Cell Sheet Technology: Scaling Up Thermo-responsive Substrates

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Vittoria Sama

Undergraduate researcher
Boston University
Boston, Massachusetts, Massachusetts, United States

Co-Author(s)

JW

Joyce Wong

Primary Investigator
Boston University
boston, Massachusetts, United States
CM

Christine McKee

PhD Candidate
Boston University, United States

Introduction: Cell sheet engineering, a scaffold-free tissue fabrication technique, has been shown to be a promising research field in regenerative medicine. Cell sheets are cell cultures grown to a high confluency form forming a monolayer with cell-produced extra-cellular matrix. The monolayer can be stacked, conditioned, and vascularized to repair and replicate native tissues. (Kino-oka1 2023)

The versatility of cell sheet technology can impact a wide range of defective tissues such as heart, skin, and cornea.

A common method for growing cell sheets is to culture cells on a thermo-responsive surface that has been grafted with poly(N-isopropylacrylamide) (PIPAAm) to control the detachment of the monolayer. (Okano 2017) Commercially available PIPAAm-grafted culture dishes are manufactured using electron beam polymerization, which requires specific equipment and is costly.

Here we focus on manufacturing a thermo-responsive surface, PIPAAM(Poly(N-isopropylacrylamide))-grafted PDMS (Polydimethylsiloxane), using UV irradiation. Previously, we have produced small, free-standing, PIPAAm-treated PDMS slabs. (Backman 2017). However, to promote accessibility, efficiency, and sustainability in this technology, we have extended this technique to accommodate a 6-well plate and any sized dish, while minimizing waste using equipment commonly found in most laboratories. Removing barriers to thermo-responsive culture dishes has the potential to help expand the range and depth of research using cell sheet technology.

Materials and

Methods: The PDMS (Polydimethylsiloxane) (The Dow Chemical Company, Midland, MI) substrate was prepared using the lid of a 6-well plate as the mold. 20% (w/v) NIPAAm (N-isopropylacrylamide) (ThermoFisher Scientific, Geel, Belgium) with 0.5% benzyl alcohol (Sigma- Aldrich, St. Louis, MO) was prepared and put in an ice bath in a desiccator at room temperature for 2 hours. The PDMS substrate was soaked in a 20% (w/v) benzophenone (Acros Organics, Ward Hill, MA) -acetone (ThermoFisher Scientific, Fair Lawn, NJ) solution for 3 minutes and rinsed with water. 0.1 % (w/v) sodium periodate solution was added to the NIPAAm solution after 2 hours. The PDMS was laid on top of the NIPAAm solution in an ice chamber during 55 mW/cm2 of UV intensity (365 nm) for 3 minutes. The PDMS was then washed with acetone first and then 200 proof 95% ethanol (Decon Labs, inc., Kind of Prussia, PA) for 3 hours each at 80°C. The PNIPAAm-PDMS substrate was dried for at least 1 hour at 80°C.

For the APTES (3 - triethoxysilylpropylamine) (Sigma- Aldrich, St. Louis, MO) treatment the PDMS was soaked in the APTES- DI water- ethanol solution for 2 hours at 80°C, and then rinsed with water for 24 hours. The surface is ready to be seeded with cells.

Results, Conclusions, and Discussions: Results
FTIR analysis of PNIPAAm- and APTES- modified PDMS substrates in well-plates shows peaks, consistent with our previous work (Backman 2017). Introducing heat during the washes reduced the processing time from 24 to 6 hours. The protocol generates minimal waste as the materials used for the PDMS mold are reusable. It is also easy to reproduce, maintaining consistent results within the previously optimized range of FTIR peak ratios (Backman 2017). Specifically, the FTIR data showed the ratio of the 1650 cm-1 amide peak (associated with pNIPAAm) to the 2900 cm-1 methyl peak (associated with PDMS) is within 0.4 to 0.5 for optimal bovine vascular smooth muscle cell (bVSMC) thermoresponsive attachment and detachment. APTES treatment helps stabilize the PNIPAAm without compromising its thermoresponsive effects as the FTIR spectra after APTES treatment shows increased peak absorbance in the broadband wavenumbers (3000–3500 cm-1) while maintaining signal in peaks corresponding to the amide bonds of the NIPAAm (1650 cm-1).

Conclusions
The process has been optimized for accessibility and sustainability by simplifying the steps and reducing needed equipment, consumables, and time. Preservation of the FTIR optimized ratio suggests successful PNIPAAM grafting for optimal bVSMC attachment/detachment. The presence of the PNIPAAm peak before and after APTES treatment suggests that the treatment did not affect the grafted PNIPAAm. We obtained repeatable results with minimal waste and by using reusable materials and equipment commonly found in the lab.

Discussions
Our redesigned procedure for generating thermoresponsive substrates addresses significant challenges in cell sheet culture by enabling researchers to manufacture their own substrates within the laboratory, allowing more scientists to conduct advanced cell culture research without relying on expensive commercial substrates. By minimizing waste, the procedure promotes more sustainable research practices. Additionally, the reproducibility of our protocol was validated with FTIR analysis. Overall, simplifying the production process and reducing costs can significantly impact cell sheet culture research, making it more accessible and sustainable for a broader range of laboratories.

Acknowledgements (Optional): We thank Dr. Jo Ann Buczek-Thomas for helpful discussions.