The present invention relates to a universal cell pairing and fusion system and method based on microfluidics, more particularly, a specific cell pairing and fusion approach in a non-mediative manner focusing on hydrodynamic induction of the cell microstructure.
In vitro artificial cell fusion is widely developed and used as an effective strategy for new cell line development, biomedical research and clinical therapies. Amounts of methods were reported on accurate and efficient cell fusion process. Most of existing studies induce target cell fusion by virtue of foreign high energy or mediations. However, these approaches are facing challenges either high dependency on accurate manipulation or destructive influences during fusion process. Moreover, many existing nuclear mechanical quantifying approaches are restricted in high facility precision and energy, which requires expensive equipment and intensive operators training.
EP0710718 A1 disclosed a method of and apparatus for cell poration and cell fusion using radiofrequency electrical pulses. The electrodes of the apparatus can be hand held or part of integrated equipment with special containers for cells.
WO2006011354 A1 disclosed a cell fusion promoter comprising ATP or its metabolite and a method of producing fused cells in the presence of ATP or its metabolite.
EP1472358 B1 disclosed a method of treating biological cells prior to subjecting the biological cells to cell fusion pulses which includes the step of treating the biological cells with pre-fusion, non-linear amplitude dielectrophoresis electric field waveforms.
However, the methods disclosed in the above patent literatures are mediative approach to artificially processing homotypic and heterotypic cell pairing and fusion which are neither accurate enough nor suitable for high-throughput operation, which cannot be realized in clinical therapy with uninjurious fusant samples.
Skelley et al. (2009) “Microfluidic Control of Cell Pairing and Fusion” reported a microfluidic device to trap and properly pair thousands of cells, compatible with both chemical and electrical fusion protocols.
Yang et al. (2016) “Optically-Induced Cell Fusion on Cell Pairing Microstructures” reported a new approach called optically-induced cell fusion (OICF), which integrates cell-pairing microstructures with an optically-induced, localized electrical field.
Rems et al. (2013) “Cell electrofusion using nanosecond electric pulses” reported a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses.
However, all methods reported in the above non-patent literatures limit in facility requirements and foreign mediation, which are not able to obtain reliable fusant without potential biotoxicity.
Pendharkar et al. (2021) “A Microfluidic Flip-Chip Combining Hydrodynamic Trapping and Gravitational Sedimentation for Cell Pairing and Fusion” reported a passive hydrodynamic cell-pairing and electrofusion strategy by using a multilayered device which is complex, lengthy in operation, and low throughput with untight cell pairing in their cell trappers. Such a device adopted electroporation to disrupt the cell membrane, which could lead to irreversible thermal injury to the cells. There were also no detailed analyses on multiple cell type fusion (homologous and heterologous parental cells) with long-term culture, sample vitality and proliferation.
A need therefore exists for an improved approach that is based on a completely hydrodynamic capture and fusion theory, e.g., micropipette-like capillary force exerted on cell membrane energy barrier during cell fusion phase, while it is electricity-free and signal control-free, to at least diminish or eliminate the disadvantages and problems described above.
Accordingly, one of the objectives of the present invention is to provide a microfluidic platform incorporating passive microtrapper arrays to realize specific cell pairing and fusion under a continuous fluid flow. At least two inlet pressure levels are applied in the microfluidic platform to drive cell samples flowing into the passive microtrappers and induce high membrane tension for cell pairing, fusion, and exchange of cellular and nuclear materials.
In a first aspect, the present invention provides a hydrodynamic cell pairing and fusion system including:
In certain embodiments, the microfluidic device includes a substrate and a microfluidic channel layer, where the microfluidic channel layer is disposed on the substrate.
In certain embodiments, the microfluidic channel layer includes a main channel, at least two fluid inlets for the main channel, at least two fluid outlets for the main channel, a cell isolation array, and a microtrapper array.
In certain embodiments, the at least two fluid inlets connect the inlet air pressure regulator.
In certain embodiments, each of the microtrappers in the microtrapper array includes at least one fluid inlet, at least two fluid outlets, and a trapping compartment, where each of the inlet and outlets communicates with different segments of the main channel in order to form a plurality of fluid bypasses.
In certain embodiments, the main channel is configured to be flexuose.
In certain embodiments, the at least one fluid inlet of each of the microtrappers is disposed more proximal to the fluid inlets of the main channel whilst the at least two fluid outlets of each of the microtrappers are disposed more distal to the fluid inlets of the main channel relative to the at least one fluid inlet of the same microtrapper.
In certain embodiments, the at least one fluid inlet of the microtrapper has a first fluid channel width; each of the fluid outlets of the microtrapper has a second fluid channel width, where the first fluid channel width is larger than the second fluid channel width.
In certain embodiments, the main channel of the microfluidic channel layer has a channel width at least sufficient for a single cell of interest to pass through with the fluid without cell rheological deformation.
In certain embodiments, the trapping compartment of each of the microtrappers has a trapper dimension (width and length) at least sufficient for capturing two cells of interest under a continuous flow of the fluid in the main channel.
In certain embodiments, the microfluidic device further includes a micropillar array for isolating cells with a size larger than the channel width of the main channel.
In certain embodiments, the main channel of the microfluidic channel layer has a channel length from where the fluid inlet of the microtrapper communicates with the main channel to where one of the fluid outlets of the same microtrapper meets with the main channel.
In certain embodiments, each of the microtrappers has a fluid channel length from the fluid inlet to one of the fluid outlets that is smaller than the channel length of the main channel.
In certain embodiments, the main channel is provided with a first fluid flow pressure at the fluid inlets of the main channel.
In certain embodiments, the first fluid flow pressure is from 0.5 to 2 psi.
In certain embodiments, the fluid outlets of each of the microtrappers has a second fluid flow pressure.
In certain embodiments, the second fluid flow pressure is from 1.0 to 10.0 kPa.
In certain embodiments, the second fluid flow pressure is higher than the first fluid flow pressure.
In certain embodiments, the first fluid flow pressure can vary to adjust the second fluid flow pressure level in the fluid outlets of the microtrapper.
In certain embodiments, the first fluid flow pressure is generated by compressed air.
In certain embodiments, the compressed air for generating the first fluid flow pressure exceeds the atmospheric pressure.
In certain embodiments, the compressed air is from the inlet pressure generator.
In certain embodiments, the microfluidic channel layer is made of a flexible and biocompatible material.
In certain embodiments, the flexible and biocompatible material for making the microfluidic channel layer includes polydimethylsiloxane (PDMS), polyvinyl alcohol (PVA), and other transparent biocompatible materials.
In certain embodiments, the substrate is made of a more rigid material than that for the microfluidic channel layer.
In certain embodiments, the more rigid material for the substrate includes glass. Polymethyl methacrylate (PMMA) and other transparent biocompatible materials with certain mechanical strength,
In certain embodiments, the surface of at least the main channel and microtrappers within the microfluidic channel layer of the present system is coated with a non-ionic, non-cytotoxic, and biocompatible surfactant to avoid cell adhesion on said surface.
In certain embodiments, the non-ionic, non-cytotoxic, and biocompatible surfactant for coating on the surface of at least the main channel and microtrappers within the microfluidic channel layer of the present system includes PLURONIC F-127 or fibronectin.
Preferably, fibronectin is coated on the surface of at least the microtrappers within the microfluidic channel layer of the present system and cell culture region, while PLURONIC F-127 is coated on the surface of the main channel.
In certain embodiments, the cells that are paired and/or fused by the present system are biological cells including animal cells, plant cells, and microorganisms, either wild-type or genetically-modified.
In certain embodiments, the cells that are paired and/or fused by the present system include immortal, tumorigenic, cancerous, pluripotent, and isogenic cells.
In a second aspect, the present invention provides a non-mediative method for pairing and fusing cells includes:
In certain embodiments, the fluid inlet of the main channel has a first flow rate and each of the fluid outlets of the microtrapper has a second flow rate, where a flow rate ratio of the second flow rate to the first flow rate is greater than 1.
In certain embodiments, the flow rate ratio of the second flow rate to the first flow rate is at least 10.
In certain embodiments, the flow rate ratio is adjusted by varying the main channel width and the fluid outlet width of the microtrapper.
In certain embodiments, said providing the hypotonic shock includes providing a hypotonic buffer for the cells in the microtrapper through the fluid inlet of the main channel and then through the inlet of the microtrapper.
In certain embodiments, the first fluid flow pressure is in a range of 0.5 to 2.0 psi.
In certain embodiments, the second fluid flow pressure is in a range of 1.0 to 10.0 kPa.
In certain embodiments, the second fluid flow pressure is in a magnitude exceeding extremum of in-plane surface tension of plasma membrane of the cells.
In certain embodiments, the second fluid flow pressure exceeding the extremum of in-plane surface tension of plasma membrane of the cells leads to cell rheological deformation in microstructure when being passively trapped into the fluid outlet of the microtrapper.
In certain embodiments, the first fluid flow pressure can vary to adjust the second fluid flow pressure level in the fluid outlets of the microtrapper.
In certain embodiments, the first fluid flow pressure is generated by compressed air.
In certain embodiments, the compressed air for generating the first fluid flow pressure exceeds the atmospheric pressure.
In certain embodiments, prior to said providing the first fluid containing the first type of cells or the second fluid containing the second type of cells to the microtrapper, the cells exceeding the width of the main channel are screened out by a micropillar array before loading to the main channel of the present system.
In certain embodiments, said monitoring cell pairing and fusion process in the trapping compartment of the microtrapper is performed by a cell pairing and fusion monitoring device of the present system including a dual-mode phase and fluorescent microscope.
In certain embodiments, after the fluid exiting the main channel, the fluid containing both fused and unfused cells is collected by a collection tube connecting to the fluid outlet of the main channel.
Other aspects of the present invention include a method of applying the present system for assessing cell pairing and fusion efficiencies of a donor or exogenous cell to a host cell. The cell pairing and fusion efficiencies obtained from the pair of donor or exogenous cell and the host cell are compared with a reference in order to evaluate the potential of the donor or exogenous cell as a therapeutic agent in a cell therapy. The present invention may also be useful in studying cell-cell interaction between more than two types of cells under different conditions.
The present invention is scalable to cope with high sample throughput demand. The flow rate, cell density, capture efficiency, and fluid pressure at various parts of the microfluidic channel layer of the present system are also adjustable to optimize the cell pairing and fusion efficiencies for a specific type of cells.
The present invention has at least the following four advantages over existing methods: firstly, the proposed cell fusion method provides potential of larger throughput product in commercial and the simplified cell capture process secures an accurate matching of parental cells to reduce random loss as traditional approaches; secondly, the fusion process is induced with cell rheological deformation in microstructure based on micropipette-inspired method, which can avoid exotic biotoxic fusogen influence; thirdly, the proposed approach would not destruct cell structures in process and cell samples can be easily recollected by driving pressures, which makes further analysis of the same cell samples become feasible; finally, fabrication of the microfluidic device is inexpensive, which can lower quantification cost. Homotypic and heterotypic cell lines are demonstrated in certain embodiments and examples described herein to verify the feasibility of the proposed artificial cell fusion approach.
Together, the present invention provides an innoxious lossless strategy for artificial cell fusion, where higher sample throughput of the proposed fusion device could be scaled up by improved parallel flux.
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Other aspects of the present invention are disclosed as illustrated by the embodiments hereinafter.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The appended drawings, where like reference numerals refer to identical or functionally similar elements, contain figures of certain embodiments to further illustrate and clarify the above and other aspects, advantages and features of the present invention. It will be appreciated that these drawings depict embodiments of the invention and are not intended to limit its scope. The invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been depicted to scale.
It will be apparent to those skilled in the art that modifications, including additions and/or substitutions, may be made without departing from the scope and spirit of the invention. Specific details may be omitted so as not to obscure the invention; however, the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation.
Turning to
Turning to
Turning to
Passive capture principle is based on continuity solution of Hagen-Poiseuille problem with the flowrate ratio between mainstream Qm and objective microtrapper Qt. The respective flowrates are derived from Darcy-Weisbach equation with determined pressure drops Δp between microtrappers and atmosphere pressure demonstrated as follows:
C(α)=96·(1−1.3553α+1.9467α2−1.7012α3+0.9564α4−0.2537α5) (2)
While the flow rate ratio Q1/Qm≥1, the inflow shows better throughput in trapper channel instead of main channel, the ratio could be determined by defined conditions following the equation (3):
To optimize capture efficiency, alternative channel dimensions (e.g., bypass width: 2 μm; main channel width: 20 μm) can be defined to afford an actual flowrate ratio much greater than 1.
Turning to
ρ(u·∇)u=∇·[−p+μ(∇u+(∇u)T)]+F (4);
and
ρ∇·(u)=0 (5)
where ρ is the density of the fluid; u is the fluid velocity; μ is the fluid dynamic viscosity; p is the fluid pressure; and F is the force contributed by the interfacial forces at the adjacent interface. Water is selected as the driven media fluid from material library and the deformed cell with a diameter of 14 μm represents a blank material with the appropriate properties defined [density=1110 kg m−3 and dynamic viscosity=0.033 Pa·s], with a refined mesh in fluid dynamics settled. The pressure drops of bypass channel are considered to be 1-10 kPa, while the cell-fluid interface is considered to be the interior wall to compute the effective surface tension for each case.
With the defined condition of cell volume conservation without irreversible membrane rupture, a geometry variation for gap height h was defined as:
The velocity profile of Poiseuille Flow could be converted into
Turning to
Where
and
for moment and moment of inertia, taking in the drag force and gap height formula, the strain energy is defined as:
Turning to
In summary, the present invention has at least the following advantages over the prior art:
Although the invention has been described in terms of certain embodiments, other embodiments apparent to those of ordinary skill in the art are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by the claims which follow.
The present invention is applicable in artificial cell fusion for in vitro biomedical research and cell therapy validation, such as cancer-stem like cells (CSCs) phenotyping in vitro model, somatic cell reprogramming, monoclonal antibodies productions, etc.