Thermo/redox-responsive dissolvable gelatin-based microsphere for efficient cell harvesting during 3D cell culturing.

The use of microspheres for culturing adherent cells has been proven as an important method, allowing for obtaining adequate number of cells in limited space and volume of medium for the intended cell-based medical applications. However, the use of proteolytic enzymes for cell harvesting from the microsphere resulted in cell damage and loss of functionality. Therefore, in this study, we developed a novel redox/thermo-responsive dissolvable gelatin-based microsphere for successful cell proliferation and harvesting adequate high-quality cells using non-enzymatic cell detachment methods. Initially, a redox-induced dissolvable gelatin-based microsphere was successfully prepared using disulfide bonds as crosslinking agent, firmly stabilizing gelatin networks and forming a stable microsphere at physiological temperature. The optimized concentration of the crosslinking agent was 1.2 mM, which kept the microsphere stable for >120 h. The microsphere was then coated with PNIPAm-ALA copolymer via physical or chemical means, resulting in a positively charged thermosensitive surface. The positive charge derived from ALA in PNIPAm-ALA copolymer enhanced cell attachment, while the thermosensitive property of the copolymer enabled for temperature induced cell harvesting. When the temperature dropped below the LCST value of PNIPAm-ALA5 (33.4°C), the copolymer swelled and became more hydrophilic, allowing cells to be readily separated. The addition of reducing agents such as GSH, DTT and L-cysteine resulted in further cleavage of the disulfide bond in the microsphere and dissolution of the microsphere for complete cell detachment. Interestingly, cell attachment and proliferation were enhanced on microspheres coated with PNIPAm-ALA5 using diselenide as a crosslinking agent, and complete cell detachment was occurred within 15 min after adding 25 mM DTT followed by lowering the temperature (4°C). Therefore, the microsphere fabricated in this study was worthwhile for non-enzymatic cell detachment and has the potential to be used for cell expansion and harvesting adequate live cells of high quality and functionality for tissue engineering or cell therapy.

Preparation of thermosensitive PNIPAm-based copolymer coated cytodex 3 microcarriers for efficient nonenzymatic cell harvesting during 3D culturing.

Enzymatic detachment of cells might damage important features and functions of cells and could affect subsequent cell-based applications. Therefore, nonenzymatic cell detachment using thermosensitive polymer matrix is necessary for maintaining cell quality after harvesting. In this study, we prepared thermosensitive PNIPAm-co-AAc-b-PS and PNIPAm-co-AAm-b-PS copolymers and low critical solution temperature (LCST) was tuned near to body temperature. Then, spin coated polymer films were prepared for cell adhesion and thermal-induced cell detachment. The alpha-step analysis and scanning electron microscope image of the films suggested that the thickness of the films depends on the molecular weight and concentration which ranged from 206 to 1330 nm for PNIPAm-co-AAc-b-PS and 97.5-497 nm for PNIPAm-co-AAm-b-PS. The contact angles of the films verified that the polymer surface was moderately hydrophilic at 37°C. Importantly, RAW264.7 cells were convincingly proliferated on the films to a confluent of >80% within 48 h and abled to detach by reducing the temperature. However, relatively more cells were grown on PNIPAm-co-AAm-b-PS (5%w/v) films and thermal-induced cell detachment was more abundant in this formulation. As a result, PNIPAm-co-AAm-b-PS (5%w/v) was further used to coat commercial cytodex 3 microcarriers for 3D cell culturing and interestingly enhanced cell detachment with preserved potential of recovery was observed at a temperature of below LCST. Thus, surface modification of microcarriers with thermosensitive PNIPAm-co-AAm-b-PS could be vital strategy for nonenzymatic cell detachment and to achieve adequate number of cells with maximum cell viability and functionality.