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, 4 (9), e6997

High-density Microwell Chip for Culture and Analysis of Stem Cells

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High-density Microwell Chip for Culture and Analysis of Stem Cells

Sara Lindström et al. PLoS One.

Abstract

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.

Conflict of interest statement

Competing Interests: Prof Helene Andersson-Svahn acts as a director, board member and has a minority stock ownership of the small startup company Picovitro AB. M. Sc. Sara Lindström has a 20% part time employment in Picovitro AB. We do not have any patents or patent applications related to this work.

Figures

Figure 1
Figure 1. Schematic overview of screening method.
(A): Cells are seeded into the microwells, either by automatic instrumentation such as flow cytometric cell-sorting (left), or manually by limited dilution (right). (B): One cell per well opens up for heterogeneity screenings, clonal assays, among other applications. Zoom in on a single cell, fixed and labeled directly after cell seeding. (C): Cell analysis, weeklong culturing and differentiation studies can be performed. Culture medium change can be performed, by rinsing the chip with fresh medium. (D): The entire chip can be screened in a rapid manner using conventional automated imaging systems, detecting cells and clones in the 672 individual wells simultaneously.
Figure 2
Figure 2. Microwell chip.
(A): Photograph of a microwell chip holding 672 microwells on a slide format of 26×76 mm. (B): Schematic drawing of one well with a volume of 500 nl, constructed as a sandwich with a glass bottom bonded to a silicon grid, creating wells. For cell culturing, a reversibly added silicone membrane is used. (C): Photograph of a well number, situated in between all wells, describing its row and column-position to enable tracking of wells for repeated imaging.
Figure 3
Figure 3. Pluripotency on chip.
(A, B): Mouse ES cells three days after plating on chip. Cells are immunoreactive to the pluripotency markers Sox2 (red) (A) and Oct4 (green) (B) and counterstained with the nuclear stain DAPI (blue). (A): Micrograph of an entire well shown in bright field (left). Close up on the colony, showing Sox2 positive ES cells, DAPI staining, and overlay of Sox2 and DAPI. (B): Close up on a colony in bright field, along with Oct4 positive ES cells, and DAPI staining. (C): Mouse adult neural stem cells three days after plating on chip. Live-cell micrograph of an entire well shown in bright-field (left). Whole-well images on Sox2 and DAPI, as well as close ups on each staining along with overlay. All micrographs were obtained using a 10× objective.
Figure 4
Figure 4. Neurospheres.
Mouse adult neural stem cells in a single-cell suspension were seeded into an uncoated chip. (A): Live cell image of an entire well showing early neurosphere formation, three days past passage. (B): Close up on a single sphere, four days past passage. Micrographs were obtained using a 10× objective.
Figure 5
Figure 5. Neural differentiation of mouse stem cells.
(A): Differentiation of ES cells under neuronal permissive conditions, nine days after plating on chip. (B): Differentiation of adult neural stem cells. Dissociated NS cells were plated and differentiated under neuronal permissive conditions for nine days on chip. Cells are immunoreactive to the neuronal marker βIII-tubulin (red) and counterstained with the nuclear stain DAPI (blue). Micrographs were obtained using a 10× objective.
Figure 6
Figure 6. Culture and neural differentiation of EBs derived from BG01 and BG01V2 hESC.
Phase contrast images of differentiating EBs derived from (A) BG01 and (B) BG01V2 illustrating that human ES cells survive well and are capable of undergoing differentiation after three days of culture in the microwells. Neuronal differentiation was confirmed by expression of the neuronal marker βIII-tubulin (green) in EBs generated from (C) BG01 and (D) BG01V2 differentiated for nine days. At this time, expression of the pluripotency marker Oct3/4 was completely lost. The cell cultures were counterstained with the nuclear stain DAPI (blue). Micrographs were obtained using a 10× or 20× objective.
Figure 7
Figure 7. Clonal assay of mouse ES cells.
Single cells were seeded into individual wells in microwell chips using flow cytometric cell-sorting, and cultured for (A) one and (B) three days. At time for analysis, the cells were visualized by staining for filamin (green), calreticulin (yellow), tubulin (red), and nucleus (blue, DAPI). (B): A clone shown with overlay of all channels. Micrographs are close ups on the cell/clone and were obtained using a 40×W objective.

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