Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Abstract

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Thermo-responsive cell culture carrier: Effects on macrophage functionality and detachment efficiency

“…Structure formulae of applied blend components are shown in Figure 1 . Additional information on physico-chemical characterization of TRP coatings by spectroscopic ellipsometry, nanoindentation, and X-ray photoelectron spectroscopy is given in Teichmann et al 18 , 19 …”

“…A high/low irradiation dose during electron beam cross-linking leads to low/high degree of swelling below the transition temperature, which corresponds to a high/low elastic modulus of the swollen material. 18 , 19 …”

“… 17 Within this context, the crucial role of cell-type-specific balance between adhesion and thermo-responsive detachment was studied to meet the requirements of particular cells and tissues. 18 , 19 These studies established a platform technology to fabricate thermo-responsive cell culture carriers with graded properties including physico-chemical and biomolecular parameters. Based on this, in this study, we focused on the characterization of various TRPs for culture and detachment of macrophages.…”

“…Based on this, in this study, we focused on the characterization of various TRPs for culture and detachment of macrophages. We systematically investigated the effects of four recently established PVME-based TRP cell culture carriers 18 , 19 and compared the results of macrophage functionality before and after cell detachment with the commercially available PNiPAAm-based cell culture carrier UpCell™ (UC; Nunc™, Thermo Fisher Scientific, Waltham, MA, USA). In particular, we used macrophages differentiated from the myeloid cell line THP-1 as well as macrophages differentiated from isolated human primary monocytes.…”

The electrospinning of a thermo-responsive polymer with peptide conjugates for phenotype support and extracellular matrix production of therapeutically relevant mammalian cells

“…Many studies to date have investigated the potential of the thermo-responsive polymer, PNIPAM, for corneal cell type detachment. 8,[35][36][37] We have recently shown that blend electrospinning of thermo-responsive polymer poly (PEGMA 188 ) with a bulk polymer of choice (amenable to electrospinning) will render the fibres thermo-responsive. Mammalian cell adhesion, viability, proliferation and phenotype on this fibrous culture system over numerous thermal enzyme-free passages was achieved.…”