Patent Application
20190344265
This invention relates to the field of biology, and more particularly, to devices for holding single cells for enabling experiments to be performed in the cells.
High-throughput screening (HTS), driven by the great progress in automation technology and combinatorial chemistry, has been widely implemented in drug discovery. As increasing considerations of earlier stage ADMET (absorption, distribution, metabolism, excretion and toxicity) in drug development, cell-based HTS is highly recommended in modern drug discovery for its ability to detect more biologically relevant characteristics of compounds in living systems.
The conventional cell-based screening is based on cell proliferation/cytotoxicity assays, monitoring the overall growth or death of a population of cells in response to treatment with specific compounds. However, conventional cell-based screening is riddled by long response times, which span from days to weeks for the whole assay time, and the possibility of interference from other intracellular pathways. In addition, current readout data from HTS are population based and does not represent the uniqueness of an individual single cell or of important sub populations, such as cancer stem cells. Since identification of the mechanism of action of a drug candidate in the cellular level is a key step stone for screening small molecules acting on therapeutic target protein or pathway, single cell observation is a key to successful drug development. Importantly, there is a lack of an effective assay method in cell-based HTS to selectively identify novel inhibitors for metastasis, though metastasis accounts for more than 90% of mortality.
Moreover, research efforts have focused on finding the molecular mechanisms underlying the bystander effect, with most studies focusing on the role of connexins and gap junctions. Treating cells and observing the behavior of adjacent cells have been the general methods used to dissect the mechanism of the bystander effect. However, researching the bystander effect at the single-cell level is a more recent trend that can uncover biological signals without the influence of uncertain elements associated with the complex structure and organization of neural networks.
To date, photoreceptor bystander research is fraught with technical challenges, including difficulties in dissociating retinal structures and quantitatively measuring bystander killing speed, unclear observations, and the lack of a reliable single-cell assay. Thus, the ability to observe and quantify the bystander killing effect in cone photoreceptors is instrumental for further mechanistic research.
To address the limitations of population based HTS and the current microinjection techniques, it would be advantageous to have a device for holding single cells separately to enable experiments on each single cell.
An aspect of some embodiments of the present invention relates to a cell holder chip, which comprises a top layer having a plurality of spaced apart microwells and a plurality of microchannels, each microwell being open on top and configured to hold a single cell, and each microwell being connected to at least two nearby microwells via respective microchannels. The microchannels are sized to enable protrusions from the cells to spread therethrough and have widths smaller than the cells to preventing the cells from entering the microchannels.
In a variant, the top layer is transparent to visible light.
In another variant, the cell holder chip further comprises a substrate bonded to a bottom of the top layer.
In yet another variant, the substrate is rigid.
In a further variant, the substrate is made of material that is transparent to visible light.
In yet a further variant, the microchannels are open on both the top and bottom of the top layer, and the substrate closes the microchannels at the bottom of the top layer.
In a variant, the cell holder chip further comprises a bottomless well plate joined to a top of the top layer, the bottomless well plate comprising a plurality of wells that traverse the plate vertically and are open both on the top and bottom of the bottomless well plate, the wells being configured to hold a liquid solution which includes cells that are to be captured in the microchannels and to enable transfer of the solution to the microwells.
In another variant, each microwell is connected to two nearby microwells via respective microchannel; or each microwell is connected to four nearby microwells via respective microchannel; or each microwell is connected to six nearby microwells via respective microchannel; or each microwell is connected to eight nearby microwells via respective microchannel.
In yet another variant, the microwells are set into an array of parallel rows, and the microchannels are straight channels connecting microwells in the same column and/or in the same row.
In a further variant, at least one of the microwells has a top cross-section has a perimeter which includes at least one inner concave angle and at least one inner convex angle, for providing increased friction between the microwell and the cell contained within.
In some embodiments of the present invention, the at least one inner convex angle is acute.
In yet a further variant, the at least one of the microwells has the top cross-section has the perimeter which includes a plurality of inner concave angles and a plurality of inner convex angle, such that a plurality of extensions are formed, protruding inward from the perimeter, to provide increased friction between the microwell and the cell contained within.
In some embodiments of the present invention, at least one of the inner convex angles is acute.
Other features and aspects of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the features in accordance with embodiments of the invention. The summary is not intended to limit the scope of the invention, which is defined solely by the claims attached hereto.
The present invention, in accordance with one or more various embodiments, is described in detail with reference to the following figures. The drawings are provided for purposes of illustration only and merely depict typical or example embodiments of the invention. These drawings are provided to facilitate the reader's understanding of the invention and shall not be considered limiting of the breadth, scope, or applicability of the invention. It should be noted that for clarity and ease of illustration these drawings are not necessarily made to scale.
Some of the figures included herein illustrate various embodiments of the invention from different viewing angles. Although the accompanying descriptive text may refer to such views as “top,” “bottom” or “side” views, such references are merely descriptive and do not imply or require that the invention be implemented or used in a particular spatial orientation unless explicitly stated otherwise.
The figures are not intended to be exhaustive or to limit the invention to the precise form disclosed. It should be understood that the invention can be practiced with modification and alteration, and that the invention be limited only by the claims and the equivalents thereof.
From time-to-time, the present invention is described herein in terms of example environments. Description in terms of these environments is provided to allow the various features and embodiments of the invention to be portrayed in the context of an exemplary application. After reading this description, it will become apparent to one of ordinary skill in the art how the invention can be implemented in different and alternative environments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this document prevails over the definition that is incorporated herein by reference.
To address the limitations and new needs for the current cell-based HTS, a chip is provided for cell protrusion-based drug screening, according to an aspect of some embodiments of the present invention. Cell protrusions are crucial components contributed to cancer cell metastasis and the cytoskeletal protrusions enable cancer metastatic colonization. In some embodiments of the present invention, the chip is configured to be integrated with standard 96-well and 384-well plates and is configured to employ microfluidics technologies to be used to screen anti-metastasis drugs detecting by high-resolution fluorescent imaging of protrusion dynamics at the single cell precision. Since the cell protrusion is very sensitive to the drug with fast response time of less than one hour, the chip of the present invention enables to identify promising drugs that inhibit or slow down cell protrusion within hours. In addition, the chip of the present invention allows to provide quantitative information about drug treatment response, cell-cell interactions, and cell-extracellular matrix interactions at single cell level in a high-throughput manner.
In some embodiments of the present invention, the chip of the present invention be used to significantly accelerate neuron research, especially the bystander effect. The chip of the present invention is capable of high-efficiency single cells capture into highly ordered microwells via a user-friendly centrifugation method and the captured single cells can be connected to adjacent cells via synapses at uniform distances along the microchannel network. Furthermore, since the chip is an up-open platform, allowing integration with a microinjection unit, the captured single cells can be quickly treated and the effects on adjacent cells can be assessed.
In some embodiments of the present invention, the microwells are configured to capture and hold single cells and to restrain the cell movement during injection. It is critically important to hold the cell as even small movement during the drug injection will punctuate large hole on the cell membrane, which can lead to cell apoptosis. For this purpose, in some embodiments of the present invention, the microwells have angled shapes and textured surface. The angle can hold cell in place without movement during injection and the textured surface provides additional fraction between cell and the wall of the microwell, preventing cell rotation and potential escape from the well during injection needle piercing cell membrane.
Traditional injection methods require to hold a cell manually with one arm and operate the injector with the other arm followed by release of the cell and move to a next target cell. In the chip of the present invention, each well contains a single cell and includes features, such as angled shape and/or walls with textured surface, which anchor the cell at the angled corner of the microwell to restrain movement during injection and increase friction between cell and the well's wall. With the chip of the present invention, the need of cell holding arm is eliminated, which makes the injection process faster and less skill dependent. In addition, the cells are isolated in pre-defined positions, which enables automation of injection and tracking of the individual cells.
Referring now to the figures,
The networked cell-holder chip 100 (hereinafter, also referred to as “chip”) includes a top layer 102 having a plurality of spaced apart microwells 104 which are open on top. Each microwell 104 has a depth d and is configured to hold an individual cell. As will be explained further below, each microwell 104 is connected to two to eight adjacent microwells via microchannels. The microchannel guide protrusions between cells in the microwells to join each other, and are smaller than the cell, therefore preventing movement of the cells through the microchannels. Only a single cell is loaded into each microwell 104. Cells spread their protrusions inside the microchannels. The microwells 104 do not pose a spatial constraint to cell morphology changes for culturing. The top layer 102 includes biocompatible material and is transparent to visible light, such as PDMS (polydimethylsiloxane), PMMA (poly (methyl methacrylate)), PC (polycarbonate), and PS (polystyrene), for example. In some embodiments of the present invention, the thickness of the top layer 102 is between 0.5 mm and 3 mm. In some embodiments of the present invention, the microwells are set into an array of parallel rows and the microchannels are straight channels connecting microwells in the same column and/or in the same row.
In some embodiments of the present invention, the top layer 102 is joined to a substrate 106. The substrate 106 is thin material that is transparent to visible light (such as glass or a rigid polymer, for example). In some embodiments of the present invention, the thickness of the substrate 106 is between 0.1 mm and 1.5 mm. The substrate 106 provides a rigid, flat base for supporting the top layer 102. The substrate 106 provides structural strength to the chip, and therefore makes the chip 100 easier to handle and move than the more compliant top layer 102.
In some embodiments of the present invention, as shown in
The top layer 102 of the chip 100 is joined to the bottom of a bottomless well plate (hereinafter, also referred to as “well plate”) 108 that is well known industry. The well plate is plate that includes a plurality of wells 100 that traverse the plate vertically and are open both on the top and bottom of the plate. The wells are configured to hold a liquid solution which includes cells that are to be captured in the microchannels and to enable transfer of the solution to the microwells. The well plate 108 is bonded with the top layer 102. The bonding may be performed by any method, such as plasma etching, adhesive bonding, or thermal bonding, for example.
In some embodiments of the present invention, at least the portion of well plate 108 which touches the solution includes biocompatible material, such as PDMS (polydimethylsiloxane), PMMA (poly (methyl methacrylate)), PC (polycarbonate), and PS (polystyrene), for example. The height H of well plate may be, for example, between 14 mm and 18 mm.
As mentioned above, each microwell 104 is configured to hold a single cell and is sized according to the cell that the microwell is configured to hold. Each microchannel 114 has a length L and a width W. The width W is smaller than the size of the cells contained in the microwells 104, to prevent the cells from escaping the microwells 104 into the microchannels 114.
In some embodiments of the present invention, the horizontal dimension D of the microwells 104 is between 10-20 μm, while the depth d of the microwells 104 (as shown in
The structure of the chip is first plotted with computer-aided design (CAD) program and then a photomask 204 is created by printing the CAD design on glass or quartz substrate. The CAD design plotted on photomask will be transferred to photoresistor layer, as will be explained further below.
In
In
The hard-baked wafer 200 and developed photoresistor 202a serve as a mold 208, shown in
In
In some embodiments of the present invention, the liquid 212 is a 10:1 mixture of a PDMS oligomer with a crosslinking prepolymer of the PDMS agent from a Sylgard™ 184 kit. The mixture is placed under vacuum for degassing, and is the poured into the basin 210 of the mold 208. The mixture is cured at 80° C. for 2 hours inside the mold 208 to assume a solid form. Once solid, the mixture is peeled off from the mold. Oxygen plasma is applied to the upper layer 102 and the thin substrate 106, and then the upper layer 102 and the thin substrate 106 are bonded together. Finally, the bottomless well plate is integrated to the bonded upper layer 102.
In
To load the cells into the chip of the present invention, a medium containing suspended cells is dropped onto the surface of the well plate of a chip of the present invention via a multichannel pipette or liquid handling system. In some embodiments of the present invention, 20-ml of the medium is dropped onto a chip of the present invention having a width of 86 mm, a length of 128 mm, and height of 14 mm. The chip is loaded on one of the receptors 302 of the bucket rotor 300. The bucket rotor is spun by the centrifuge 304 to drive the cells into the microwells of the chip. The inventors have experimented with different spinning modes. A non-limiting spinning mode that worked well for introducing 661 W cells into the microwells of the chip is as follows:
(i) 0 to 1000 rpm, acceleration=1
(ii) 1000 rpm for 1 min;
(iii) 1000 to 600 rpm, deceleration=1;
(iv) 600 to 0 rpm, deacceleration=9.
After spinning, the chip 100 is transferred to an inverted microscope 306, such EVOS FL Auto 2microscope manufactured by Thermo Fisher Scientific. Filter papers (e.g., manufactured by Whatman) are used to swipe away the unloaded cells. Finally, the cells are cultured at 37° C. in the chip 100 a humidified atmosphere with 5% CO2, to enable protrusions to extend in the microchannels. The cells are also stained (for example, with green fluorescent protein—GFP) to enhance visibility of the cells and protrusions.
The graph of
In some embodiments of the present invention, the microwells 104 in the chip have walls are shaped to increase friction between the microwell and the cell contained within. This friction decreases the movement of the cell within the microwells and therefore facilitates microinjection in the cells.
In some embodiments of the present invention, each microwell, has a top cross-section having a perimeter which includes one or more inner concave angles and one or more inner convex angles. In some embodiments of the present invention, at least one of the convex angles is an acute angle. These shapes of the top cross-section are determined by the shape of the photomask used to fabricate the chip. In some embodiments of the present invention, the perimeter of the top cross section has a plurality of inner concave angles and inner convex angles, such that a plurality of extensions protrude inwards from the perimeter, as seen, for example in
In
This application claims priority from U.S. Provisional Application Ser. No. 62/668,606 filed May 8, 2018, which is hereby incorporated herein by reference in the respective in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62668606 | May 2018 | US |
