Dissociation of brain tissue into viable single neurons in a microfluidic device
01 Nov 2015-pp 29-32
TL;DR: A microfluidic technology-based tissue-dissociation device has for the first time been designed, fabricated and characterized for the purpose of primary neuronal cell culture of freshly explanted, enzyme-treated Drosophila larval central nervous system into individual, viable neurons capable of robust outgrowth during in vitro culture.
Abstract: A microfluidic technology-based tissue-dissociation device has for the first time been designed, fabricated and characterized for the purpose of primary neuronal cell culture. The system has been utilized for controlled dissociation, under an oscillatory flow field, of freshly explanted, enzyme-treated Drosophila larval central nervous system (CNS) into individual, viable neurons capable of robust outgrowth during in vitro culture. Device dimensions, constriction height and width, and operating conditions, flow-rate amplitude and frequency, have been determined based on video microscopy as well as quantitative analyses of the subsequent neuron-culture results.
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TL;DR: In this article, a microfluidic platform that can dissociate native biological tissue into ready-to-use single cells was proposed, which can merge the successive steps of tissue dissociation, debris filtration, cell sieving, washing, and staining in one streamlined process.
Abstract: The advancement of sample preparation techniques is essential for the field of cell-based therapeutics. To obtain cells suited for clinical applications, the entire process starting from acquiring donor tissue biopsy, all through cell transplantation into the recipient, should occur in an integrated, safe, and efficient system. The current laboratory approach for solid tissue-to-cell isolation is invasive and involves multiple incoherent manual procedures running in an open operator-dependent system. Such an approach provides a chain of events for systematic cell loss that would be unfavorable for rare cell populations such as adult and cancer stem cells. A few lab-on-chip platforms were proposed to process biological tissues, however, they were limited to partial tissue dissociation and required additional processing off-chip. Here, we report the first microfluidic platform that can dissociate native biological tissue into ready-to-use single cells. The platform can merge the successive steps of tissue dissociation, debris filtration, cell sieving, washing, and staining in one streamlined process. Performance of the platform was tested with diverse biological tissues and it could yield viable cells that were ready for on or off-chip cell culture without further processing. Microfluidic tissue dissociation using this platform produced a higher number of viable single cells (an average of 2262 cells/ml per milligram of tissue compared to 1233.25 cells/ml/mg with conventional dissociation).
4 citations
TL;DR: A proof‐of‐concept fluid shear‐based mechanical dissociator that was designed to dissociate stem cell spheroids and aggregates was tested and had the potential to replace traditional dissociation methods.
Abstract: Biological industries commonly rely on bioreactor systems for the large-scale production of cells. Cell aggregation, clumping, and spheroid morphology of certain suspension cells make their large-scale culture challenging. Growing stem cells as spheroids is indispensable to retain their stemness, but large spheroids (>500 µm diameter) suffer from poor oxygen and nutrient diffusion, ultimately resulting in premature cell death in the centers of the spheroids. Despite this, most large-scale bioprocesses do not have an efficient method for dissociating cells into single cells, but rely on costly enzymatic dissociation techniques. Therefore, we tested a proof-of-concept fluid shear-based mechanical dissociator that was designed to dissociate stem cell spheroids and aggregates. Our prototype was able to dissociate cells while retaining high viability and low levels of apoptosis. The dissociator also did not impact long-term cell growth or spheroid formation. Thus, the dissociator introduced here has the potential to replace traditional dissociation methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:293-298, 2018.
1 citations
References
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TL;DR: It is shown that the compensation system can counteract the effects of extremely unnatural environmental conditions—a temperature step—in which the anterior and posterior halves of the embryo are developing at different temperatures and thus at different rates.
Abstract: Biochemical networks are perturbed both by fluctuations in environmental conditions and genetic variation. These perturbations must be compensated for, especially when they occur during embryonic pattern formation. Complex chemical reaction networks displaying spatiotemporal dynamics have been controlled and understood by perturbing their environment in space and time. Here, we apply this approach using microfluidics to investigate the robust network in Drosophila melanogaster that compensates for variation in the Bicoid morphogen gradient. We show that the compensation system can counteract the effects of extremely unnatural environmental conditions-a temperature step-in which the anterior and posterior halves of the embryo are developing at different temperatures and thus at different rates. Embryonic patterning was normal under this condition, suggesting that a simple reciprocal gradient system is not the mechanism of compensation. Time-specific reversals of the temperature step narrowed down the critical period for compensation to between 65 and 100 min after onset of embryonic development. The microfluidic technology used here may prove useful to future studies, as it allows spatial and temporal regulation of embryonic development.
448 citations
"Dissociation of brain tissue into v..." refers background in this paper
...These techniques are compatible with living cells, allowing for unprecedented ability to control the cellular microenvironment in culture [8]....
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TL;DR: Four applications of a miniaturized, disposable microbial culture chip suggest that the potential for such simple, readily manufactured chips to impact microbial culture is extensive and may facilitate the full automation and multiplexing of microbial culturing, screening, counting, and selection.
Abstract: A miniaturized, disposable microbial culture chip has been fabricated by microengineering a highly porous ceramic sheet with up to one million growth compartments. This versatile culture format, with discrete compartments as small as 7 x 7 mu m, allowed the growth of segregated microbial samples at an unprecedented density. The chip has been used for four complementary applications in microbiology. (i) As a fast viable counting system that showed a dynamic range of over 10,000, a low degree of bias, and a high culturing efficiency. (ii) In high-throughput screening, with the recovery of 1 fluorescent microcolonly in 10,000. (iii) In screening for an enzyme-based, non-dominant phenotype by the targeted recovery of Escherichia coli transformed with the plasmid pUC18, based on expression of the lacZ reporter gene without anti biotic-resistance selection. The ease of rapid, successive changes in the environment of the organisms on the chip, needed for detection of beta-galactosidase activity, highlights an advantageous feature that was also used to screen a metagenomic library for the same activity. (iv) In high-throughput screening of >200,000 isolates from Rhine water based on metabolism of a fluorogenic organophosphate compound, resulting in the recovery of 22 microcolonies with the desired phenotype. These isolates were predicted, on the basis of rRNA sequence, to include six new species. These four applications suggest that the potential for such simple readily manufactured chips to impact microbial culture is extensive and may facilitate the full automation and multiplexing of microbial culturing, screening, counting, and selection.
266 citations
"Dissociation of brain tissue into v..." refers background in this paper
...Microfluidic systems have recently been reported for high-throughput screens and studies of biological species such as bacteria [9]....
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TL;DR: This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system axon myelination research and shows excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels.
Abstract: This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16-18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.
168 citations
"Dissociation of brain tissue into v..." refers methods in this paper
...Microfluidic devices have been used to separate and culture manually dissociated neurons [10-14]....
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TL;DR: Although many intricate microfluidic devices have been created in academic laboratories around the world, far fewer have been commercialized for wider use as mentioned in this paper. But several efforts are underway to bridge this divide.
Abstract: Although many intricate microfluidic devices have been created in academic laboratories around the world, far fewer have been commercialized for wider use. But several efforts are underway to bridge this divide.
78 citations
"Dissociation of brain tissue into v..." refers background in this paper
...New processes such as soft lithography and selfassembly monolayers (SAMs) allow the fabrication of microscale devices with feature sizes ranging between 1 m and 1cm [7]....
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TL;DR: Key features of various LTP induction protocols in dissociated hippocampal neuronal cultures and the applications of these plasticity models for the investigation of activity-induced changes in native AMPA receptors are focused on.
77 citations
"Dissociation of brain tissue into v..." refers background in this paper
...Primary neuron culture is a particularly powerful tool for revealing the regulation of neuronal development, including neurogenesis; survival; interactions with other cells; as well as synapse formation and plasticity [2]....
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