Researchers in the US have devised a no-contact measurement technique that can monitor the viability of live cells in a bioprinted tissue construct. Current measurement approaches largely rely on destructive analysis of the synthesized structures by staining and sectioning, while in-process optical imaging tools only provide information on the size and shape of the biofabricated construct.
The new technique, developed by a team of researchers at North Carolina State University, assesses the critical quality attributes (CQAs) of a bioprinted construct by measuring the dielectric impedance of the cells contained within it. This approach can be used to determine the total number of live cells, how these cells are distributed, and even the state they are in. “The work could also help bring bioprinting a step closer to automation, which will bring down costs,” explains Binil Starly, who led this research effort together with Rohan Shirwaiker.
The technique exploits the inherent dielectric properties of living cells, which arise from their double-shell structure that consists of the cell membrane and enclosed cytoplasm. When an alternating electric field is applied, positive and negative charges build up across the membrane, making the cell behave as a capacitor with a permittivity that depends on the frequency of the electric field. This interfacial polarization, which is known as the Maxwell-Wagner effect, is not seen in non-viable cells, which often have ruptured membranes.
“When we apply an alternating current, the cells within the 3D construct either allow the flow of current through the cells, or impede it,” explains Starly. “If the cells are healthy with intact membranes, it strongly impedes the flow. If they are damaged – after having passed through the nozzle of a bioprinter, for example – or stressed by having been in the printer for a long time, the cells are not strong enough to impede the current and allow electrons to pass right through.”
Measuring without destroying
The measurements require no direct contact with the construct, do not cause any damage to the cells, and do not need any fluorescent dyes. “Based on how the cells within the construct behave within this AC field, we can tell how many viable cells there are and assess how many live cells are distributed within a large construct,” explains Starly. “And, even if all the cells are viable, we can also determine whether the state of the construct is different to how it should be.”
While the current study measured cell viability just after the construct had been printed, Starly believes that the technique could integrated into a bioprinter to enable real-time characterization of the biofabricated constructs. Such an approach would have a clear advantage over current in-process metrology tools, which rely on video cameras to take optical images of the construct at regular intervals as it forms.
“While determining feature dimensions in this way is important for measuring how functional the construct is, it does not provide any information on the state of the cells contained within it, either during printing or immediately after the printing process has ended,” says Starly. “Our in-process measurement tool allows us to monitor the biofabrication process and actively control it to produce robust 3D tissue constructs.” he adds.
Probe needs to be modified
The researchers, reporting their results in the IOP journal Biofabrication, tested their technique on human adipose-derived stem cells in 3D hydrogel constructs, and they now want to study how the measurement signals change when there are two or more cell types within a construct. “For example, how could we deconstruct the signal to identify characteristics specific to a particular cell type?” asks Starly.
From a technical point of view, the researchers also plan to modify the dielectric impedance spectroscopy probe in their bioprinter. “The probe used in this study was never designed for such a context but rather for use in large bioreactors and fermenters in the beer brewing industry, in which there are much larger numbers of cells than in our system,” says Starly.
The team, which includes students Lokesh Narayanan and Trevor Thompson, says it also needs to measure the permittivity of the cells within a more focused frequency range of between 150 and 2500 kHz, relevant to mammalian cells. “At present, a frequency scan last for 30 seconds and we obtain permittivity readings across 24 discrete frequencies between 50 and 20 000 kHz, of which only 11 are relevant,” says Starly.
- Read our special collection “Frontiers in biofabrication” to learn more about the latest advances in tissue engineering. This article is one of a series of reports highlighting high-impact research published in Biofabrication.