Researchers report on the development of a microfluidic platform that allows the long-term culture and study of stem cell proliferation at the single-cell level. They claim the nanoliter chamber-based array system essentially mimics conventional culture systems, and allows the precise, automated control of medium exchange without disturbing the cells, so their responses to changing conditions can be evaluated by live-cell imaging.
Studies by the University of British Columbia-led team confirmed that mouse hematopoietic stem cells cultured using the system subsequently retained their functional properties in vitro and in vivo. The researchers describe their approach, and in vitro and in vivo assay results, in Nature Methods. The paper is titled “High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.”
Although integrated microfluidic systems are used for the study of yeast and bacterial cell responses to various stimuli, scientists have struggled to develop equivalent systems suitable for the long-term study of biological responses in mammalian cells, notes Carl L. Hansen, Ph.D., at UCB’s Centre for High-Throughput Biology, and colleagues. Technical hurdles to achieving this goal include overcoming cell dehydration, the immobilization of nonadherent cells during medium exchange, and the recovery of cultured cells for subsequent phenotypic or functional analysis. As a result, mammalian microfluidic culture systems to date have been restricted in the main to experiments with adherent cells, incubated for a maximum of a few hours in relatively large volumes of media, and maintained under high perfusion rates.
Dr. Hansen’s team has now developed a microfluidic cell culture platform it claims solves these problems and supports growth rates that replicate standard macrocultures. Constructed from polydimethylsiloxane (PDMS), the platform features 1,600 cell culture chambers with just over a 4 nL volume each, with integrated microvalves for the precise control and exchange of medium. PDMS is permeable to some small molecules and water vapor, however, and this can cause a change in medium composition, or dehydration in high surface area-to-volume nanoliter-sized culture chambers, leading to spurious biological responses, reduced growth rates or cell death, the researchers point out. To circumvent this problem they designed their culture system such that the chambers are surrounded by an iso-osmotic medium-filled bath to increases both humidity and the overall volume-to-surface ratio.
A critical feature of the design is the arrangement of the flow channels, which run over the tops of the chambers. A peristaltic pump is used to pass the initial cell culture across the tops of the chambers at a rate that transfers cells over every chamber, but without causing a stress response. Only once the pump is stopped do the cells drop into the chambers, where they are then essentially trapped by gravity. The process can be repeated to achieve a more concentrated loading. Medium exchange through the array can then be carried out at a flow rate that doesn’t disturb the cells in their chambers.
Importantly, the device allows for cell recovery either in bulk, or selectively from individual chambers using a micropipette. For the bulk method the array is inverted and flushed through, which allows about 90% cell recovery, the authors claim. The micropipetting method also allows for more than 90% recovery of cells from a selected chamber.
To test the system Dr. Hansen’s team first compared the growth rates of preleukemic mouse cells cultured in the microfluidic apparatus with those of cells growth in conventional cultures. In the presence of the iso-osmotic bath the microarray-cultured cells doubled at a rate equivalent to that of cells grown in 24-well plates and in 96-well single cell cultures. In contrast, in devices that lacked the iso-osmotic bath, cell survival and division were both severely compromised, despite humidity control and the initiation of medium exchanges 24 hours after starting the experiment, the researchers note.
Culturing normal primary mouse hematopoietic stem cells (HSCs) for five days confirmed that the cell division kinetics of HSCs grown in the microfluidic device were comparable over successive divisions to those of cells cultured using macroscale systems, and resulted in high cell concentrations. In fact, at confluence (about 150 cells per chamber), the system exceeded the limits of cell concentration that can be sustained in conventional batch cultures, they claim.
Primitive hematopoietic cells grown in the microfluidic system and subsequently transplanted into mice were also found to retain their function. In this experiment NA10hd-transduced adult mouse bone marrow cells expanded in macroscale culture were transferred to either the microfluidic array or a 96-well-plate macroscale system and cultured for an additional 60 hours.
The resulting cultured cells from both systems were subsequently transplanted into irradiated mice together with bone marrow helper cells. All mice showed similar reconstitution levels by the transplanted cells for more than 16 weeks post-transplantation, suggestive of an overall HSC expansion of more than 600-fold, the authors report. Reconstitution of both the myeloid and lymphoid compartments was also equivalent for both sources of expanded HSCs.
Using an enlarged, 6,144 chamber system, the researchers were subsequently able to investigate the effects of low steel factor concentration on HSC survival and proliferation. Previous work has suggested that low SF concentrations cause rapid loss of HSC function, delayed proliferation and increased death, while subsequent high concentrations of the cytokine can rescue the cells. However, what hasn’t been clear to date, they note, is how long quiescent adult HSCs could be exposed to low SF concentrations before subsequent rescue by high concentration of SF was no longer possible. Their studies with primary mouse HSCs suggested that the cells could be rescued from declining viability within the first 16 hours of culture with low SF concentration. After that, high concentrations of SF couldn’t reverse the damage.
The microfludic system is ideal for the long-term live-cell imaging studies of clonal cultures of nonadherent cells, the researchers conclude. And while not unique to microfluidic systems, the throughput and automation of the system should allow for detailed studies of colony growth and variability that would otherwise be impractical.” “Our device has several features that are applicable to investigate heterogeneous populations of mammalian cells that have stringent medium requirements. These features include large number of chambers, nonperturbing cell immobilization, dynamic medium exchange, and robust long-term cell culture ... The ability to replace the culture medium without disturbing cells is essential to avoid nutrient limitations that occur in longer-term experiments, especially for small culture volumes where even a few cells rapidly grow to high concentrations.”
Dr. Hansen et al. suggest their technology could have widespread utility for applications spanning drug-response screens, culture optimization, clone selection, and cell characterization. “This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.”