On-chip clearing of arrays of 3-D cell cultures and micro-tissues

Abstract

Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 lm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear T2 , and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear T2 ; however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This onchip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. Published by AIP Publishing. [http://dx

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

“…The results demonstrated that the method induces a minimal reduction of the size of the spheroids, that is, the ratio of spheroids area before and after the clearing was 1.02 (Grist et al, 2016). The results demonstrated that the method induces a minimal reduction of the size of the spheroids, that is, the ratio of spheroids area before and after the clearing was 1.02 (Grist et al, 2016).…”

“…The results demonstrated that the method induces a minimal reduction of the size of the spheroids, that is, the ratio of spheroids area before and after the clearing was 1.02 (Grist et al, 2016). An analysis of the average FUCCI fluorescence intensity as a function of penetration depth into the sample demonstrated that the SeeDB method allowed to image the spheroids at depths greater than 250 μm, while the imaging of noncleared spheroid was limited to about 150 μm (Grist et al, 2016). The transparency was slightly improved since the light transmittance increased to about 5.5% in the cleared spheroids (Grist et al, 2016).…”

“…This reduction is dependent on the flow rate used in the microfluidic channel, that is, higher flow rates lead to higher shrinkage, which may be related to the fluidic compressive forces or to the osmotic pressure. Nevertheless, the authors also observed that SeeDB solution increased the green fluorescence intensity of the spheroids (Grist et al, 2016), which was attributed to the formation of Maillard reaction products (Berke, Miola, David, Smith, & Price, 2016 occurred as a consequence of the clearing process (Boutin & Hoffman-Kim, 2015). An analysis of the average FUCCI fluorescence intensity as a function of penetration depth into the sample demonstrated that the SeeDB method allowed to image the spheroids at depths greater than 250 μm, while the imaging of noncleared spheroid was limited to about 150 μm (Grist et al, 2016).…”

“…These methods aim to reduce the light-scattering phenomenon in the biological samples, rendering samples with increased transparency that promotes deeper imaging, as well as the cells structural and functional analysis (discussed hereafter). In this way, the light penetration is usually limited to a range of 100-150 μm, which is insufficient for the imaging of the spheroids whole volume (Grist, Nasseri, Poon, Roskelley, & Cheung, 2016;Richardson & Lichtman, 2015). This phenomenon promotes light scattering and it is the main cause of tissues opacity.…”

Simulation-assisted design of microfluidic sample traps for optimal trapping and culture of non-adherent single cells, tissues, and spheroids

“…The trapping mechanisms to load samples within the device vary depending on the design (Fig. 1A) and can combine many mechanisms studied by different authors, such as i) sedimentation trapping, where waiting for the tissue to settle into an extrusion from a channel traps it 7–10 ; ii) resistive trapping, which exploits preferential flow to guide samples into traps 11–16 ; iii) inertial trapping, which uses focusing flows or sharp turns to trap samples in channel recesses 17–19 ; iv) dielectrophoretic trapping, where the difference in permittivity between the fluid and tissue is exploited to trap it in an electric field 20, 21 ; v) and open-microfluidic channel networks, which create hanging droplets in which samples are trapped or synthesized 22, 23 .

Figure 1Basic MST designs: ( A ) MST operation with schematics of different trapping mechanisms: resistive, dielectrophoretic, inertial and sedimentation trapping; blue axis is parallel to gravity. The picture of the western hemisphere of earth on a transparent background was solely created by NASA and is in the public domain in the United States.

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