A comparative analysis of detachment forces and energies in initial and mature cell-material interaction

Single cell force spectroscopy (SCFS) enables data on interaction forces to be acquired during the very early adhesion phase. However, SCFS detachment forces and energies have not been compared so far with the forces and energies after maturation of the cell-material contact on a single cell level and with comparable time resolution. We used FluidFM (R) to physically attach single cells to the cantilever by aspiration through a microfluidic channel, in order to achieve the higher forces required for detaching maturely adhering cells. Combining these two approaches allowed us to compare cell adhesion in the initial and maturation phases of adhesion for two exemplary cell-substrate combinations - L929 fibroblasts on fibronectin and MC3T3 osteoblasts on collagen type I. Uncoated glass substrates were used as a reference. For both cell lines, SCFS measurements after contact times of 5, 15 and 30 s revealed significantly higher maximum detachment forces (MDFs) and energies on glass compared to the protein-coated surfaces in the 0.5-4 nN (1-40 fJ) range. FluidFM (R) measurements after 1, 2 and 3 days of culture revealed a significant absolute increase in the MDFs and detachment energies for both cell lines on protein-coated substrates to values of about 600 nN and 10 pJ. On glass, the MDFs were similar for MC3T3 cells, while they were significantly lower for L929 cells. For both cell types, the differences in detachment energy were significant. These differences underline the importance of investigating early and mature adhesion states to obtain a holistic assessment of the cell-material interactions.The authors are grateful to the DFG (German Research Council) graduate school GRK1505/2 "Welisa" and grant number BA 2479/2-1 for funding the position of P. Wysotzki, as well as the consumables for the experiments. We also acknowledge the ERC (European Research Council), Grant Number 617989 for the financial support given. We are grateful to Dr. W. Baumann (Department of Biophysics, Univ. of Rostock) for helpful discussions

Entwicklung von Zellkultursystemen mit integrierter Sensorik und Aktuatorik für die Überwachung von Knochenzellkulturen

Ziel war die Entwicklung eines Zellkultursystems mit Mikrofluidik zur Messung zellphysiologischer Parameter wie Ansäuerungsrate, Sauerstoff-verbrauch sowie Zelladhäsion. Durch den Einsatz von Mikrosensoren soll die metabolische Aktivität sowie das Proliferationsverhalten der Zellen untersucht werden. Das System beeinhaltet interdigitale Elektrodenstrukturen (IDES) zum Nachweis der Zelladhäsion, pH- und Sauerstoffsensoren zur Bestimmung von Ansäuerungsrate und Sauerstoffverbrauch. Es erfolgten Zellmessungen mit MG-63 und MC3T3-E1 Zelllinien

Fast Prototyping of Sensorized Cell Culture Chips and Microfluidic Systems with Ultrashort Laser Pulses

We developed a confined microfluidic cell culture system with a bottom plate made of a microscopic slide with planar platinum sensors for the measurement of acidification, oxygen consumption, and cell adhesion. The slides were commercial slides with indium tin oxide (ITO) plating or were prepared from platinum sputtering (100 nm) onto a 10-nm titanium adhesion layer. Direct processing of the sensor structures (approximately three minutes per chip) by an ultrashort pulse laser facilitated the production of the prototypes. pH-sensitive areas were produced by the sputtering of 60-nm Si3N4 through a simple mask made from a circuit board material. The system body and polydimethylsiloxane (PDMS) molding forms for the microfluidic structures were manufactured by micromilling using a printed circuit board (PCB) milling machine for circuit boards. The microfluidic structure was finally imprinted in PDMS. Our approach avoided the use of photolithographic techniques and enabled fast and cost-efficient prototyping of the systems. Alternatively, the direct production of metallic, ceramic or polymeric molding tools was tested. The use of ultrashort pulse lasers improved the precision of the structures and avoided any contact of the final structures with toxic chemicals and possible adverse effects for the cell culture in lab-on-a-chip systems