Summary Microscale techniques have been applied to biological assays for nearly two decades, but haven’t been widely integrated as common tools in biological laboratories. The significant differences between several physical phenomena at the microscale versus the macroscale have been exploited to provide a variety of new types of assays (such as gradient production or spatial cell patterning). However, the use of these devices by biologists seems to be limited by issues regarding biological validation, ease of use, and the limited available readouts for assays done using microtechnology. Critical validation work has been done recently that highlights the current challenges for microfluidic methods and suggest ways in which future devices might be improved to better integrate with biological assays. With more validation and improved designs, microscale techniques hold immense promise as a platform to study aspects of cell biology that are not possible using current macroscale techniques.

We have developed a technique for fabricating microfluidic devices from gelatin using a natural crosslinking process. Gelatin, crosslinked with the naturally occuring enzyme transglutaminase is molded to produce microchannels suitable for adherent cell culture and analysis. The autofluorescence of the material was shown to be minimal and within the range of typical background, ensuring utility with analyses using fluorescent dyes and labels would not be affected. Also, normal murine mammary epithelial cells were successfully cultured in the microchannels. The morphology of these adherent epithelial cells was shown to be significantly different for cells grown on rigid tissue culture plastic in either macro-or microscale cultures (even in the presence of a surface coating of gelatin) than those grown on the flexible crosslinked gelatin microchannels. Using these devices, the effects of both the extracellular matrix and soluble factors on cellular behavior and differentiation can be studied in microenvironments that more closely mimic the in vivo environment. IntroductionWhile microfluidics has shown considerable promise as a tool for studying cell biology, the potential for microfluidics to create more in vivo-like in vitro environments (or, in visio environments) is still largely untapped. It is becoming clear that the scale of the microenvironment provided by microchannels is an important biological parameter. Microchannels have been used for several steps in the in vitro production of embryos typically either matching or improving the performance of previous methods. 1,2 Insect cell cultures as well have shown very different dimension dependent growth kinetics in microscale cultures as compared to macroscale flask cultures. 3 However, much of the previous culture work in microfluidics has focused on non-adherent (e.g., embryos, insect cells) or 2-D adherent cell culture in the absence of extracellular matrix. While the effects of the extracellular matrix are of critical importance to in vivo tissue function, 4,5 the integration of extracellular matrix (ECM) materials into microfluidic systems is just emerging. 6,7 We have fabricated microdevices which can allow for control over the soluble microenvironment, while also providing a more in vivo-like substrate (flexible protein gels) as a step towards an in visio environment for cellular analysis. We present a fabrication protocol to produce enzymatically crosslinked gelatin microdevices composed of natural components, that are sterile and suitable for cell culture. Gelatin is a derivative of collagen, one of the most common extracellular matrix proteins. These devices were tested for their fluorescence properties to ensure that any autofluorescence of the material itself will not limit its utility while using fluorescent dyes for cellular analysis. Also, we have explored the potential for mammary epithelial cell culture in these devices and compared the cell morphology at multiple time points between macro-and microscale cultures on traditional rigid surfaces an...

Genomic studies have revealed significant branching heterogeneity in cancer. Studies of resistance to tyrosine kinase inhibitor therapy have not fully reflected this heterogeneity because resistance in individual patients has been ascribed to largely mutually exclusive on-target or off-target mechanisms in which tumors either retain dependency on the target oncogene or subvert it through a parallel pathway. Using targeted sequencing from single cells and colonies from patient samples, we demonstrate tremendous clonal diversity in the majority of acute myeloid leukemia (AML) patients with activating internal tandem duplication mutations at the time of acquired resistance to the FLT3 inhibitor quizartinib. These findings establish that clinical resistance to quizartinib is highly complex and reflects the underlying clonal heterogeneity of AML.

Clonal evolution in cancer – the selection for and emergence of increasingly malignant clones during progression and therapy, resulting in cancer metastasis and relapse – has been highlighted as an important phenomenon in the biology of leukemia and other cancers. Tracking mutant alleles to determine clonality from diagnosis to relapse, or primary site to metastases, in a sensitive and quantitative manner is most often performed using next generation sequencing. Such methods determine clonal frequencies by extrapolation of allele frequencies in sequencing data of DNA from the metagenome of bulk tumor samples using a set of assumptions. The computational framework that is usually employed assumes specific patterns in the order of acquisition of unique mutational events and heterozygosity of mutations in single cells. However, these assumptions are not accurate for all mutant loci in acute myeloid leukemia (AML) samples. In order to assess whether current models of clonal diversity within individual AML samples are appropriate for common mutations, we developed protocols to directly genotype AML single cells. Single cell analysis demonstrates that mutations of FLT3 and NPM1 occur in both homozygous and heterozygous states, distributed among at least 9 distinct clonal populations in all samples analyzed. There appears to be convergent evolution and differential evolutionary trajectories for cells containing mutations at different loci. This work suggests an underlying tumor heterogeneity beyond what is currently understood in AML, which may be important in the development of therapeutic approaches to eliminate leukemic cell burden and control clonal evolution-induced relapse.

Microfluidic devices for cell culture based assays provide new types of engineered microenvironments and new methods for controlling and quantifying cellular responses to these microenvironments. However, without an understanding of the effects of the microenvironments present in microdevices from a cellular perspective, it will be challenging to integrate work done in microdevices with biological data obtained via traditional methods. With the adaptation and validation of In Cell Westerns (ICWs) and in situ analysis techniques to microfluidic devices, we can begin to look at a variety of cellular responses to microcultures. Here we observe several differences in proliferation, glucose metabolism, signaling pathway activation and protein expression levels between cells cultured in traditional macroscale cultures and in microfluidic cultures. The issues of glucose starvation, growth factor restriction, volume density and effects of interactions with poly(dimethylsiloxane) (PDMS) were examined to determine the relative importance of each to cell behavior. Changes in glucose metabolism, insensitivity to volume density or media supplementation, and finally reduced proliferation as the exposure to PDMS increased, suggests that perhaps interactions between media/cells and this commonly employed polymer may be significant for some cell based assays. The differences between cells in macroscale and microfluidic cultures suggest that the cellular baseline may be substantially altered in microcultures due to both inherent differences in scale as well as material differences. The observations highlight the need to biologically validate micofluidic devices for cell based assays in order to accurately interpret the data obtained with them in the context of traditional macroculture data. Additional areas of study that will further characterize and validate microscale culture are discussed.