Microfluidic platform for 3D cell culture with live imaging and clone retrieval

Mulas, Carla; Hodgson, Andrew Christopher; Kohler, Timo N.; Agley, Chibeza C.; Humphreys, Peter; Kleine‐Brüggeney, Hans; Hollfelder, Florian; Smith, Austin; Chalut, Kevin J.

doi:10.1039/d0lc00165a

Cited by 20 publications

(25 citation statements)

References 38 publications

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“…We encapsulated hPSCs in agarose microgels to investigate epiblast morphogenesis in a chemically defined 3D scaffold ( Kleine-Brüggeney et al., 2019 ; Mulas et al., 2020 ) ( Figure 1 A). Primed hPSCs cultured in self-renewing conditions (Essential 8 medium [E8]) ( Chen et al., 2011 ) were dissociated to single-cell suspension and mixed with a low-melting-point agarose solution.…”

Section: Resultsmentioning

confidence: 99%

“…Microfluidic devices have been extensively used for the generation of monodisperse hydrogel microdroplets to compartmentalize biological and chemical reactions ( Fischlechner et al., 2014 ; Huebner et al, 2007 , 2008 ; Theberge et al., 2010 ). Recent applications include the encapsulation of dissociated mouse PSCs in agarose microgels to combine live imaging with precise cell retrieval for endpoint analysis ( Kleine-Brüggeney et al., 2019 ; Mulas et al., 2020 ). Microgels provide a scalable platform from which to study PSCs in the context of a synthetic 3D matrix and represent an exciting avenue to combine embryonic and extraembryonic lineages for stem cell-based embryo models.…”

Section: Introductionmentioning

confidence: 99%

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Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells

Schindler¹,

Siriwardena

Kohler³

et al. 2021

Stem Cell Reports

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Human periimplantation development requires the transformation of the naive pluripotent epiblast into a polarized epithelium. Lumenogenesis plays a critical role in this process, as the epiblast undergoes rosette formation and lumen expansion to form the amniotic cavity. Here, we present a high-throughput in vitro model for epiblast morphogenesis. We established a microfluidic workflow to encapsulate human pluripotent stem cells (hPSCs) into monodisperse agarose microgels. Strikingly, hPSCs self-organized into polarized epiblast spheroids that could be maintained in self-renewing and differentiating conditions. Encapsulated primed hPSCs required Rho-associated kinase inhibition, in contrast to naive hPSCs. We applied microgel suspension culture to examine the lumen-forming capacity of hPSCs and reveal an increase in lumenogenesis during the naive-to-primed transition. Finally, we demonstrate the feasibility of co-encapsulating cell types across different lineages and species. Our work provides a foundation for stem cell-based embryo models to interrogate the critical components of human epiblast self-organization and morphogenesis.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells

Schindler¹,

Siriwardena

Kohler³

et al. 2021

Stem Cell Reports

Self Cite

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“…Microfluidic platforms are a good option if one needs to combine live imaging with retrieving individual cells of interest [93,316].…”

Section: Image Detection and Recognition Techniquesmentioning

confidence: 99%

Cultivating Multidisciplinarity: Manufacturing and Sensing Challenges in Cultured Meat Production

Djisalov

Knežić

Podunavac

et al. 2021

Biology

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Meat cultivation via cellular agriculture holds great promise as a method for future food production. In theory, it is an ideal way of meat production, humane to the animals and sustainable for the environment, while keeping the same taste and nutritional values as traditional meat and having additional benefits such as controlled fat content and absence of antibiotics and hormones used in the traditional meat industry. However, in practice, there is still a number of challenges, such as those associated with the upscale of cultured meat (CM). CM food safety monitoring is a necessary factor when envisioning both the regulatory compliance and consumer acceptance. To achieve this, a multidisciplinary approach is necessary. This includes extensive development of the sensitive and specific analytical devices i.e., sensors to enable reliable food safety monitoring throughout the whole future food supply chain. In addition, advanced monitoring options can help in the further optimization of the meat cultivation which may reduce the currently still high costs of production. This review presents an overview of the sensor monitoring options for the most relevant parameters of importance for meat cultivation. Examples of the various types of sensors that can potentially be used in CM production are provided and the options for their integration into bioreactors, as well as suggestions on further improvements and more advanced integration approaches. In favor of the multidisciplinary approach, we also include an overview of the bioreactor types, scaffolding options as well as imaging techniques relevant for CM research. Furthermore, we briefly present the current status of the CM research and related regulation, societal aspects and challenges to its upscaling and commercialization.

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“…The use of imaging is an essential requirement for the study of the structural and functional morphology of the neo-organ, of its positioning / polarization and of cell differentiation, allowing the in vitro modeling of even the most complex organs [31]. Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge [80]- [82].…”

Section: Microfluidic Live Imagingmentioning

confidence: 99%

“…Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes [80]. Technological advances have enabled a broad array of live-cell imaging approaches to investigate cellular processes, many of which require optically demanding imaging modalities to achieve the necessary resolution and sensitivity [83].…”

Section: Microfluidic Live Imagingmentioning

confidence: 99%

Microfluidic Live-Imaging technology to perform research activities in 3D models

Capuzzo

Vigo

2021

Preprint

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One of the most surprising differences observed when comparing cell cultures in 2D and 3D is morphological dissimilarity and their evolution over time. Cells grown in a monolayer tend to flatten in the lower part of the plate adhering to and spreading in the horizontal plane without expanding in the vertical dimension. The result is that cells grown in 2D have a forced apex-basal polarity. 3D cultures support co-cultivation and crosstalking between multiple cell types, which regulate development and formation in the in vivo counterpart. 3D models culture, with or without a scaffold matrix, can exhibit more in vivo-like morphology and physiology. 3D cultures recapitulate relevant physiological cellular processes, transforming into unique platforms for drug screening. To support and guarantee the functional maintenance of a 3D structure, one must consider the structures and dynamics of regulatory networks, increasingly studied with live-imaging microscopy. However, commercially available technologies that can be used for current laboratory needs are limited, although there is a need to facilitate the acquisition of cellular kinetics with a high spatial and temporal resolution, to elevate visual performance and consequently that of experimentation. The CELLviewer is a newly conceived and developed multi-technology instrumentation, combining and synchronizing the work of different scientific disciplines. This work aims to test the system with two models: the first model is a single Jurkat cell while the second is an MCF-7 spheroid. After having grown both models, the two models used are loaded into the microfluidic cartridge for each experiment and recorded in time-lapse for a total of 4 hours. After adaptive autofocus, when sliding inside the cartridge chamber, the samples used are tracked under the action of the optics and the 3D rotation was experimentally successfully obtained. A cell viability assessment was then used using the MitoGreen dye, a fluorescence marker selectively permeable to live cells. The ImageJ software was used to: calculate the model diameter, create fluorescence intensity graphs along a straight line passing through the cell, visualize the spatial fluorescence intensity distribution in 3D.

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Microfluidic platform for 3D cell culture with live imaging and clone retrieval

Abstract: 3D cell culture and microfluidic platform for monitoring biological process and single clone retrieval for downstream molecular or functional analysis.

Cited by 20 publications

References 38 publications

Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells

Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells

Cultivating Multidisciplinarity: Manufacturing and Sensing Challenges in Cultured Meat Production

Microfluidic Live-Imaging technology to perform research activities in 3D models

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