Single Cell Deposition Research Articles

Directed self-assembly software for single cell deposition.

Laser direct-write (LDW) bioprinting methods offer a diverse set of tools to design experiments, fabricate tissue constructs and to cellular microenvironments all in a CAD/CAM manner. To date, we have just scratched the surface of the system’s potential and for LDW to be utilized to its fullest, there are many distinct hardware and software components that must be integrated and communicate seamlessly. In this perspective article, we detail the development of novel graphical user interface (GUI) software to improve LDW capability and functionality. The main modules in the control software correspond to cell transfer, microbead fabrication, and micromachining. The modules make the control of each of these features, and the management of printing programs that utilize one or more features, to be facile. The software also addresses problems related to construct scale-up, print speed, experimental conditions, and management of sensor data. The control software and possibilities for integrated sensor data are presented.

Simplifying stable CHO cell line generation with high probability of monoclonality by using microfluidic dispensing as an alternative to fluorescence activated cell sorting.

Single cell cloning is a critical step for cell line development (CLD) for therapeutic protein production, with proof of monoclonality being compulsorily sought in regulatory filings. Among the different single cell deposition technologies, we found that fluorescence activated cell sorting (FACS) offers high probability of monoclonality and can allow selective enrichment of the producer cells. However, FACS instruments are expensive and resource-intensive, have a large footprint, require highly skilled operators and take hours for setup, thereby complicating the cell line generation process. With the aim of finding an easy-to-use alternative to FACS, we identified a flow cytometry-based microfluidic cell dispenser, which presents a single cell sorting solution for biopharmaceutical CLD. The microfluidic cell dispenser is small, budget-friendly, easy-to-use, requires lower-cost consumables, permits flow cytometry-enabled multiparametric target cell enrichment and offers fast and gentle single cell dispensing into multiwell plates. Following comprehensive evaluation, we found that single cell deposition by the microfluidic cell dispenser resulted in >99% probability of monoclonality for production cell lines. Moreover, the clonally derived producer cell lines generated from the microfluidic cell dispenser demonstrated comparable or improved growth profiles and production capability compared to the FACS derived cell lines. Taken together, microfluidic cell dispensing can serve as a cost-effective, efficient and convenient alternative to FACS, simplifying the biopharmaceutical CLD platform with significant reductions in both scientist time and running costs.

Abstract LB161: Single cell bioprinted cell circuits for the systematic assessment of cell-cell communication in the early tumor microenvironment

Abstract The specific communication of multiple cell types in the tumor microenvironment plays a critical role in cancer progression. Current engineering methods have failed to adequately replicate the complexities of the tumor microenvironment (TME). In particular, generating engineered tissue-like environments with multiple TME cell types has remained challenging. Here we demonstrate the capability to pattern complex single cell circuit configurations, using a novel microfluidic bioprinting method, to study cell-cell communication in the early TME. A microfluidic dispenser (Biopixlar, Fluicell AB) was optimized to determine the delivery pressure (5 – 80 mbar), internal vacuum (0 – 80 -mbar), and external vacuum (0 – 80 -mbar) to enable highly controllable deposition of single cells suspended in complete media supplemented with polyethylene glycol (15 mg/mL, 1:1) at 1 × 106 cells/mL. Flow conditions were optimized for human cells: MDA-MB-231, MCF7, PC3, breast epithelial cells (MCF10a), fibroblasts, cancer associated fibroblasts, THP-1 derived macrophages, CD4+ T cells, CD8+ T cells, human umbilical vein endothelial cells (HUVECs), and mesenchymal stem cells. As proof of concept, the optimized settings were used to replicate a 2D tumor biopsy region of interest with high spatial precision. Next, cell-cell communication circuits were fabricated with cancer cells (PC3 or MDA-MB-231) and HUVECs. Communication circuits were bioprinted as 4 by 4 cell arrays, with 100 µm spacing between each cell, equal number of HUVECs and cancer cells, and three different cellular arrangements: alternating cell types, like cell types grouped, and groups of four like cell types. The circuits were live cell imaged for up to 30 hours to observe cell migration patterns, proliferation, and morphological changes as a function of cell-cell communication circuit arrangements. Optimal printing parameters were identified as 80 mbar delivery pressure, -25 mbar internal vacuum, and -55 mbar external vacuum. These parameters maintained >99% cell viability and ±10 µm spatial precision of printed cells. Live cell imaging of circuits containing PC3s or MDA-MB-231s with HUVECs on collagen substrates revealed changes in migration patterns, proliferation, and morphology depending on the surrounding cellular arrangement. HUVECs were highly migratory throughout the duration of the experiment, frequently extended protrusions towards nearby HUVECs, but did not display the same level of interaction with PC3s as they did with MDA-MB-231s. In MDA-MB-231 circuits, irrespective of patterning, we identified clear tendencies of HUVECs to herd MDA-MB-231s, travel overtop of MDA-MB-231s, collect and carry visible particles released from MDA-MB-231s, and maintain dendritic morphology instead of undergoing the expected vascular tubulogenesis. We found that HUVECs had the best morphology when clustered in groups of four and proliferated most when surrounded by MDA-MB-231s (alternating pattern). We found that MDA-MB-231s only proliferated when surrounded by HUVECs and had the least displacement when surrounded by like cells. These results demonstrate a method to precisely bioprint single cell circuits, enabling the investigation of cellular spatial organization and composition within the tumor microenvironment as it relates to tumor initiation and progression. Citation Format: Haylie R. Helms, Alexander E. Davies, Rebekka Duhen, Joshua M. Moreau, Ellen M. Langer, Luiz E. Bertassoni. Single cell bioprinted cell circuits for the systematic assessment of cell-cell communication in the early tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB161.

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters.

Characterization of a novel microfluidic platform for the isolation of rare single cells to enable CTC analysis from head and neck squamous cell carcinoma patients.

Detailed examination of tumor components is leading‐edge to establish personalized cancer therapy. Accompanying research on cell‐free DNA, the cell count of circulating tumor cells (CTCs) in patient blood is seen as a crucial prognostic factor. The potential of CTC analysis is further not limited to the determination of the overall survival rate but sheds light on understanding inter‐ and intratumoral heterogeneity. In this regard, commercial CTC isolation devices combining an efficient enrichment of rare cells with a droplet deposition of single cells for downstream analysis are highly appreciated. The Liquid biopsy platform CTCelect was developed to realize a fully‐automated enrichment and single cell dispensing of CTCs from whole blood without pre‐processing. We characterized each process step with two different carcinoma cell lines demonstrating up to 87 % enrichment (n = 10) with EpCAM coupled immunomagnetic beads, 73 % optical detection and dispensing efficiency (n = 5). 40 to 56.7 % of cells were recovered after complete isolation from 7.5 ml untreated whole blood (n = 6). In this study, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR‐based confirmation of tumor‐related biomarkers and immunostaining. Finally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. This system offers new possibilities for single cell screening in cancer diagnostics, individual therapy approaches and real‐time monitoring.

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

Coupling picodroplet microfluidics with plate imaging for the rapid creation of biomanufacturing suitable cell lines with high probability and improved multi-step assurance of monoclonality.

There is an expectation from regulatory agencies that cell lines used in the commercial production of biopharmaceuticals are derived from a single cell progenitor. Traditional methods of single cell cloning include the use of the limiting dilution cloning method which often requires multiple rounds of low cell density cell plating and either microscopic evaluation that wells contain single cells and/or the calculation of a statistically derived probability of monoclonality. We have combined the single cell screening, deposition and picodroplet imaging ability of Sphere Fluidics' Cyto-Mine technology with the plate imaging capability of the Solentim Cell Metric to create a novel workflow for the generation of high producing clonal cell lines with both high probability and assurance of monoclonality. The efficiency of three key stages of the process (single cell picodroplet encapsulation, single picodroplet dispensation and single cell settling in the focal plane of the plate imager) was determined and a probability calculation was derived using the Wilson Score Interval method. The combined probability that a single cell is encapsulated into a picodroplet, is deposited into the correct well of a 96-well plate and that a cell settles into the focal plane of the plate imager yields a combined>99% probability of monoclonality. Furthermore, visual verification of a single cell progenitor is obtained at multiple steps throughout the cloning workflow. This novel methodology for the rapid creation of high quality clonal cell lines for biomanufacturing purposes has many advantages over more traditional approaches including improved assurance of single cell derivation, integrated imaging capability, assay flexibility, equipment utilization time and in-process cell line segregation.

Methods for Estimating the Probability of Clonality in Cell Line Development.

Mammalian cell banks for biopharmaceutical production are usually derived from a single progenitor cell. Different methods to estimate the probability that the cell banks are clonally derived, or the probability of clonality (PoC), associated with various cloning workflows have been reported previously. In this review, a systematic analysis and comparison of the methods used to calculate the PoC are provided. As the single cell deposition and high-resolution imaging technologies continue to advance and the cloning workflow evolves, an aligned understanding and best practice on estimating the PoC is necessary to compare different cloning workflows adopted across the biopharmaceutical industry and it will help to accelerate regulatory acceptance.

Integration of silver nanowires into SU-8 hollow cantilevers for piezoresistive-based sensing

We established a process for the microfabrication of SU-8 AFM cantilevers with an embedded microchannel and an integrated layer of silver nanowires as piezoresistive element. The self-sensing detection is based on the electrical resistance increase of the silver nanowire network associated with the strain applied upon deflection of the cantilever. We observed a sensitivity of 1.4 × 10−6 nm−1 at large strains of 4 % (gauge factor of 10) and a sensitivity of 0.1 × 10−6 nm−1 at small strains of 0.6 % (gauge factor of 1). We obtained a still higher sensitivity (gauge factor of 28) by coating the silver nanowires network with a thin (∼10 nm) aluminum oxide layer acting as an insulating barrier. As application, we studied the cantilever deformation as a function of the overpressure applied in the microchannel while the cantilever was kept in contact with a glass surface confirming their large deflection detection capabilities. The results were qualitatively rationalized in the percolation framework. The presented self-sensing micropipettes could be used for experiments that require remarkably important bending, such as localized single cell deposition by squeezing the micropipette against the substrate, as well as force-controlled electrochemical additive manufacturing at the sub-mm scale.

Mitochondrial membrane potential identifies cells with high recombinant protein productivity

Development of cell lines for biotherapeutic protein production requires screening large numbers of clones to identify and isolate high producing ones. As such, stable cell line generation is a time- and resource-intensive process. There is an increasing need to enhance the selection efficiency of high-yielding clonal cell lines for cell line development projects by using high throughput screening of live cells for markers predictive of productivity. Single cell deposition by fluorescence activated cell sorting (FACS) is a commonly performed method for cloning to generate cell lines derived from a single recombinant cell. We have developed a novel strategy to identify higher productivity cells at the FACS step by leveraging a simple viable cell staining method that detects mitochondrial membrane potential (Ψm), a key indicator of cellular metabolic activity. We chose a dual-emission dye (Mito-ID, Enzo Life Sciences) that fluoresces green and orange in living cells with the intensity of the orange fluorescence being dependent on the cells Ψm status. Using available clonal cell lines with known productivity, or stable transfectant pools, we evaluated Ψm of cell populations with Mito-ID dye. We determined that the intensity of the Ψm fluorescent signal correlates with the known fed-batch titers of the producer clones, and that cell sorting based on an optimal Ψm staining intensity selectively enriches for higher producing clones from nonclonal transfectant pools. These clones are phenotypically stable for recombinant protein production. Furthermore, the strategy has been successfully applied to identifying higher producing cell lines for a range of antibody molecular formats. Using this method, we can combine an enriching step with the cloning step for high producers, thereby saving time and resources in cell line development.

Surfaces functionalized by graphene oxide nanosheets for single cell investigations

In this research development of sensor suitable for single cell investigation is reported. To achieve this goal biocompatible graphene oxide (GO) nanosheets were applied as substrates for separated cell immobilization. GO nanosheets are particularly interesting due to their biocompatibility, scalability, and possibility to be deposited via simple surface functionalization protocols. In this context, we rise the hypothesis that GO nanosheets can be used to treat substrates for single cell studies. We investigated this hypothesis using several pristine and GO-modified highly oriented pyrolytic graphite, objective glass slides, polished silicon wafers, polydimethylsiloxane and mica substrates. A significantly larger frequency of single cell deposition events was observed on all substrates modified by GO. We exploited the GO functionalization to advance the Raman spectroscopy based analysis of the chemical composition of individual cells gathering different Raman spectra with respect to that collected from yeast cell colonies.

A single-step FACS sorting strategy in conjunction with fluorescent vital dye imaging efficiently assures clonality of biopharmaceutical production cell lines.

The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation. Within the flow cytometry step, two unit operations are evaluated separately, doublet discrimination during event selection for deposition and droplet deposition accuracy. The imaging procedure is evaluated for the accuracy of detection of non-clonal populations. By employing mixing experiments of cell populations, the authors demonstrate that doublet discrimination is highly efficient, and that an appropriately set up flow cytometry system already can generate >99.5% true single cell clones. The efficiency of the described imaging process depends on several factors, reaching an optimal detection rate of non-clonal wells of about 99.8%. Our results demonstrate that one well characterized cloning step generate biopharmaceutical production cell lines with a probability of clonality of >99.99%.

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies.

The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten-horseradish peroxidase conjugate (hapten-HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG-Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.

Design and analysis of new fault-tolerant majority gate for quantum-dot cellular automata

Due to its ultrasmall size and extremely low power consumption, quantum-dot cellular automata (QCA) technology represents a promising alternative to semiconductor transistors at the nanoscale. Nevertheless, the design of QCA circuits is limited by their high defect rate during fabrication, making fault-tolerant QCA structures a popular research topic. The aim of this work is to design a new fault-tolerant QCA majority gate based on a $$3\times 5$$3×5 tile. The majority gate guarantees good fault tolerance under single cell and double cell missing defects compared with several previous structures. The functional results when adopting the polarization and kink energy of the proposed majority gate under such single cell and double cell deposition defects are fully investigated. Besides, a series of new fault-tolerant adders are implemented based on the fault-tolerant majority gates. To evaluate the performance of the proposed adders, a thorough comparison versus previous adders with respect to fault tolerance, cell number, area, and delay is carried out. The results indicate that the proposed design can reach a high level of fault tolerance, and perform rather well in terms of other properties. For simulation analysis, the QCADesigner tool is used to check the functionality of all circuits.

Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.

The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

A rapid method to verify single-cell deposition setup for cell sorters.

Studying single cells reveals biology that cannot be explored using bulk techniques. Cell sorters provide the opportunity of separating single cells either for cell culture or for downstream molecular applications such as qPCR to study specific gene expression and single cell mRNA sequencing. Some of these molecular studies can be expensive so the investigator will often want reassurance that the cell sorter can reliably deposit a single droplet into each well of a 96-well or 384-well plate. Such plates may contain very small volumes of fluid as reducing the volume of fluid used can reduce the cost of the assay. To miss some of the wells could leave the data set incomplete requiring costly repetition. To verify this by microscopy is at best very time consuming and at worst impossible. Here, an inexpensive colorimetric method is described for verifying whether a well, in either a 96- or 384-well plate, did receive a single sorted droplet from a cell sorter into the fluid at the bottom of the well. The droplet consists of particles suspended in an enzymatic solution, horseradish peroxidase, which is deposited into microtiter plate wells containing a substrate, 3,3',5,5'-tetramethylbenzidine. This method requires no special equipment or expertise and is rapid enough to be performed directly prior to the single-cell sorting experiment. © 2016 International Society for Advancement of Cytometry.

Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.

Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.

Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins.

Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging.

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low‐density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 31:1172–1178, 2015

Automated single cell sorting and deposition in submicroliter drops

Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4–0.7 μl with a sorting speed of 15–20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.