One of the HTE Program missions is to perform a scientific and technical monitoring. So, some weeks ago, we had the opportunity to follow partners from our HetColi network in Zurich, where we met a physicians team of IBM Research. We have discovered the microfluidic based technologies developed in the Govind Kaigala team, called Microfluidic Probes (MFP).

Govind, a former hardward design engineer, performed a PhD and a Master Enginering in Canada, then a Post-doc in Stanford, USA (Figure 1 : Picture adapted from IBM Research content). He always developed a double competence, working in engineering and medical biology. He was hire at IBM Research in 2010 and found a very productive team. Govind team developed Microfluidic Probe (MFP), a technology inspired by scanning technologies from IBM that can interact with biological supports like cells or tissues.

They proposed a non-contact platform that can hydro- dynamically localize very small volumes of liquid on a surface, at the µmeter level. As seen on Figure 2, they connected a microfluidic pumping system to a self designed and made MFP head. Basically, the MFP head consist in a tiny tip containing two apertures/µchannels, one for injection of a processing liquid and one for aspiration of the liquid. MFP head is put very close to the sample in an immersion liquid. The scientist could control the flow and create a hydrodynamic confinement upside the sample and perform a spatio-temporal alteration of surfaces. This equipment can measure until 175 × 100 × 140 mm3 and scan an area of 45 × 45 mm2 on a surface with an accuracy of ±15 μm. So it could be placed on a microscope for use on histological slides, microtiter plates, or Petri dishes.

The probes could be more complex than in the Figure 1. Indeed, the majority of their probes have more than 2 apertures, and can inject and aspire a shaping liquid allowing a finest confinment of the processing liquid (Figure 3). Each probe is designed for a specific application. Indeed, MFP could have a broad range of applications based on its ability to perform a local analysis. Govind lab developed a lot of strategies to customize throughput of biological samples in term of histology, selective lysis, DNA, RNA, protein analysis and so one.

The first example is the use of MFP for retrospective analysis of tissue sections from histological slides and biobanks embedded in FFPE. Usually you take a histological slide and dewax the whole sample in order to perform histological or molecular analysis. With MFP it is possible to dewax a very restricted area and performed a molecular analysis without alteration of the surrounding tissue sample (Figure 4). The video 1 also illustrates this ability. Indeed, by using xylene as processing liquid it is possible to realize a microscale dewaxing and shape a unique pattern of dewaxing by moving the MFP head at µmeter level. In a second time, a classical histological staining or a miniaturized immunochemistry could be performed.

Video 1. Microscale dewaxing

The scanning ability of the MFP platform allowed to design a lot of staining pattern. As shown on Figure 5, it is possible to shape patterns like dots, lines, contourings, gradients and so one. In the same way, immuno-histochemistry (IHC) could be performed according the same miniaturized level. Figure 6illustrates that point with some examples in breast cancer histological slides. Indeed, Govind team show us a dot staining of p53 with MFP technology. They also develop developed “multiplex” staining in very close area with p53 and progesterone receptors antibodies. This miniaturisation and the ability to perform kind of antibodies multiplexing is of interest with tissue micro array (Figure 6).

Moreover, according to physicochemical properties, tiny volumes and a constant flow of fresh antibodies in the processing liquid, hybridization times are very shorter than in classical IHC protocol. This point is one of the big advantages of MFP technology transforming classical technologies with faster and cheaper protocols.

Govind lab also adapted MFP also to DNA/RNA in situ hybridization (Figure 7). Indeed they developed a miniaturized version of fluorescence in situ hybridization (FISH), logically named µFISH. One more time, volumes miniaturization and allowed a localized increase in DNA or RNA probe concentration. Again, the consequence is a reduction in probe consumption and a lower incubation time.

Moreover, they shown they can perform a “spatially multiplexed µFISH“ by hybridization of 2 different probes on two sample area very closely localized, as seen on Figure 7. Another exciting use of MFP is the ability to perform a spatially resolved lysis of biological substrate for downstream analysis. Indeed, the lysate could be analyzed at DNA or RNA level for example. They can collect from 30 to 100 cells very localized. The video 2 illustrates this selective lysis on a MCF7 cell line culture.

Video 2. Selective lysis

This targeted lysis was done on patients biopsies as shown in the Figure 8. Govind team extracts DNA from a trans thoracic lung biopsy with adenocarcinoma and search for mutation of BRAF gene. To lyse from 30 to 100 cells, it only need a 15 seconds passage over the histological slide. Collected cells could also be used for RNA expression studies. This targeted lysis property could also be very useful as a tool to create in vitro heterogeneity in cell culture. For exemple, on a monocellular layer, lysis of cells according to a define shape followed by the seeding of a different cell line will create a personalized heterogeneous coculture. Other interesting applications could be adapted from MFP technology (very localized temperature variations or shear stress, biopatterning,…). To explore more about the work of this physicians team, just have a look at their publications on IBM website or PubMed.

In conclusion, MFP technology seems to be a new class of analytical methods at the micro-scale level. It is adaptable to a broad range of molecular analysis currently used by scientists or clinicians. To summarize the advantages of MFP, we could say that this technology is sample, time and cost-saving. Its use for deciphering the solid tumors heterogeneity is a promising avenue.

Bibliography:

1) Convection-Enhanced Biopatterning with Recirculation of Hydrodynamically Confined Nanoliter Volumes of Reagents. Autebert J, Cors JF, Taylor DP, Kaigala GV. Anal Chem. 2016 Mar 15;88(6):3235-42. doi: 10.1021/acs.analchem.5b04649. Epub 2016 Feb 22.

2) Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections. Cors JF, Kashyap A, Fomitcheva Khartchenko A, Schraml P, Kaigala GV. PLoS One. 2017 May 11;12(5):e0176691. doi: 10.1371/journal.pone.0176691. eCollection 2017.

3) A compact and versatile microfluidic probe for local processing of tissue sections and biological specimens. Cors JF, Lovchik RD, Delamarche E, Kaigala GV. Rev Sci Instrum. 2014 Mar;85(3):034301. doi: 10.1063/1.4866976.

4) Micro-immunohistochemistry using a microfluidic probe. Lovchik RD, Kaigala GV, Georgiadis M, Delamarche E. Lab Chip. 2012 Mar 21;12(6):1040-3. doi: 10.1039/c2lc21016a. Epub 2012 Jan 12.

5) Micro fluorescence in situ hybridization (μFISH) for spatially multiplexed analysis of a cell monolayer. Huber D, Autebert J, Kaigala GV. Biomed Microdevices. 2016 Apr;18(2):40. doi: 10.1007/s10544-016-0064-0.

6) Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe. Kashyap A, Autebert J, Delamarche E, Kaigala GV. Sci Rep. 2016 Jul 14;6:29579. doi: 10.1038/srep29579.

7) Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe.Kashyap A, Cors JF, Lovchik RD, Kaigala GV. J Vis Exp. 2016 Sep 15;(115). doi: 10.3791/54447.