KR20120013012A - Techniques for isolation of axons from cultured neurons and formation of directional synapses between neurons - Google Patents

Abstract

PURPOSE: A method for separating spatial axons from neurons is provided to manufacture a bio chip which enables signal recognition of neuron of a low density compartment. CONSTITUTION: A method for separating spatial axon comprises: a step of selectively injecting first suspension liquid containing neurons into a first injection hole to attach the neurons to a neuron adhesion layer; a step of culturing the attached neurons; and a step of isolating axons extended from the neurons. A microfluid chip comprises a first flow path having the first injection hole and a first discharge hole; a second flow path having a second injection hole and a second discharge hole; a microfluid channel for connecting the first and second flow paths; and a neuron adhesion layer.

Description

Techniques for isolation of axons from cultured neurons and formation of directional synapses between neurons

The present invention relates to a method of synapse formation having a spatial axon separation and directionality from nerve cells, and more specifically, axon projection in one direction of low density neurons in high density neurons using a microfluidic chip for axon separation. The present invention relates to a method for separating spatial axons that grow and separate into microfluidic channels.

In vitro culture of biological tissues or cells extracted from the animals without direct use has made a significant contribution to the development of research in cell biology and medicine. Various researches on tissue culture and cell culture through primary culture and subculture have been conducted, and the protocol for various kinds of cells is very well established.

Despite efforts to observe environmental changes around individual neurons of neurons, low density cultures are very difficult because neurons grow by interacting with cultured neurons.

Until now, it has been studied that in order to obtain a reasonable neuronal viability for conducting the study, the neurons must be cultured to a certain density or more (10 to 50 cell / mm 2). Moreover, general culture conditions (tissue culture dish, tissue culture flask, tissue culture plate) are very difficult to bring about the change of population per unit area because neurons are injected at a constant density (in a single density).

Therefore, the nerve cells of interest are always crowded with so many bumps (axons and dendrites) that it is very confusing to identify their origins and relationships.

On the other hand, in recent years, analytical researches on the behavior of neurons according to environmental changes around individual neurons have been actively conducted. To do this, individual neurons must be isolated from the neuronal cell population, isolated and cultured, sufficient space must be provided around the neuron, the influence from surrounding neuronal cell populations must be controlled, and axons isolated from the neuronal cell population. The protuberance must be cultured inducing around isolated neurons.

In particular, in the past studies, the separation of axons from cell bodies has been carried out using separation techniques using materials such as nerve cell growth factor (NGF).

Therefore, the present inventors do not need to use materials such as nerve growth factor (NGF), photolithography process, polymer replica molding technique, soft lithography of semiconductor processing technology. Microfluidic chip for axon projection separation, and combined with surface patterning method, a new method of spatial axon projection in which axons grow in one direction from low density neurons to microfluidic channels. The separation method was found and the present invention was completed.

An object of the present invention is to spatially isolate a small number of nerve cells from the injected whole nerve cell group, and then culture for a predetermined time, and then to spatially separate the axon projections around the isolated nerve cells, and to the isolated axons and isolated nerve cells. It is intended to provide a method of forming a directional synapse around a cell.

Still another object of the present invention is to provide a method for forming an artificial synapse having directivity between axons separated by the above separation method and neurons grown at low density.

In order to achieve the object as described above, the present invention uses a pattern formation and the microstructure on the surface of the microfluidic chip for separation of axons, through the control of the density of nerve cell culture according to the position on the same substrate In one direction to the low-density neurons in the axon growth is separated into a microfluidic channel, to provide a separation method of spatial axon projections and separated axon projections through the separation method.

In the separation method of the axon protrusion of the present invention, by using the microfluidic chip for separating the axon protrusion, nerve cells containing different concentrations of nerve cells are injected through the first injection hole and the second injection hole on the same substrate, and the nerve cells are injected. It is characterized in that the adhesion of the spatially selective neurons to the adhesion layer is made.

Separation method of the axon projection of the present invention is characterized in that by using the microfluidic chip for separation of the axon projections, nerve cells grow at different injection densities in the neuronal cell adhesion layers of the first flow path and the second flow path on the same substrate. .

Separation method of the axon projections of the present invention is characterized in that the axon projections grow in one direction of the low density neurons from the high density neurons by using the microfluidic chip for separation of the axon projections.

The microfluidic chip for separating the axon protrusions of the present invention connects the first flow path having the first inlet and the first outlet, the second flow path having the second inlet and the second outlet, and the first and second flow paths. It is characterized in that the microfluidic chip for separation of the axon projections comprising a microfluidic channel, the first flow path, the second flow path and the neuron cell adhesion layer provided on the base surface of the microfluidic channel.

Hereinafter, the present invention will be described in detail.

The present invention

A first flow passage having a first inlet and a first outlet, a second flow passage having a second inlet and a second outlet, a microfluidic channel connecting the first and second flow passages, the first flow passage, and a second flow passage In the axon projection separation method using a microfluidic chip for axon projection separation comprising a nerve cell adhesion layer provided on the base surface of the microfluidic channel,

Selectively injecting a first suspension containing neurons into the first inlet and attaching to the neuronal cell adhesion layer; And

Separating and growing axons in the microfluidic channel through the culture of the attached nerve cells;

It provides a separation method of spatial axon projections from nerve cells using a microfluidic chip for separation of axon projections and provides axon projections of nerve cells separated by the method of the axon projections.

In the present invention, the separation method of the axon projections is characterized in that the second suspension injected through the second inlet is a suspension containing nerve cells of lower density than the first suspension.

The first and second suspensions injected through the first and second inlets are suspensions containing neurons of different densities, and more specifically, suspensions containing high density neurons through the first inlets. Or a suspension containing neurons of high density through the first inlet and a suspension containing neurons of low density through the second inlet.

In the present invention, the nerve cells attached to the neural cell adhesion layer of the first flow passage or the second flow passage are separated by a concentration gradient through the microfluidic channel in one direction of high density to low density.

In the present invention, the nerve cells attached to the specific part can be separated in two dimensions in one direction of high density to low density, and only axons and dendrites grow and move by concentration gradient through the microfluidic channel. Are separated.

In the separation method of the spatial axon projection of the present invention, the first injection hole and the second injection hole are injected with a suspension containing nerve cells of different densities, and the injected nerve cells are attached and grown at different injection densities, In order to induce growth and separation of axons with spatially selective neuronal attachment and orientation, surface patterning of neuronal cell attachment materials, or the installation of three-dimensional microstructures, or microfluidic chips with many compartments By using the compartment and the microfluidic channel connecting the compartments, the axons can be separated in a predetermined direction by controlling the direction as each protrusion grows and controlling the growth rate.

In more detail, physical separation according to the growth rate is used to separate only the axons from the cell body in a specific direction. The axons have a growth rate of 100 to 200 μm / day and a growth rate of 20 to 30 μm / day. If the dendrite grows competitively toward the vacant space in addition to the space to which the neurons are attached, only the axons separated from the cell body can move and separate through the microfluidic channel after a certain period of time.

In the present invention, the microfluidic chip is one or more polymers or light selected from polydimethylsiloxane (PDMS), polyurethane (PU), polymethyl methacrylate (PMMA) on a substrate By attaching a microstructure made of a sensitive polymer to form a flow path and a microfluidic channel.

In the present invention, the neuron adhesion layer is selected from glass, silicon, silicon oxide (silicon oxide), silicon nitride (silicon nitride), polycarbonate (polycarbonate), polystyrene (polystyrene) and polypropylene (polypropylene) 1 It is characterized by being located on more than one substrate.

In addition, the neuron adhesion layer simulates poly-L-lysine (PLL), poly-D-lysine (PDL), laminin, laminin structure One or more neuronal cell attachment substances selected from one synthetic polypeptide or oligopeptide can be used.

More specifically, the structure of poly-L-lysine (PLL), poly-D-lysine (PDL), laminin, laminin structure on the substrate is simulated. One or more neuronal cell attachment substances selected from synthetic polypeptides or oligopeptides are printed using a polymer such as polydimethylsiloxane (PDMS) to form a pattern in advance and grow neurons thereon, with cell adhesion It created an environment that can only grow on matter.

In the present invention, the flow passage has a section of 100 to 1000 ㎛, length 1 to 20 mm, height 50 to 500 ㎛, characterized in that connected to the microfluidic channel, the microfluidic channel is 3 to 100 width It is characterized in that the plurality of microfluidic channels spaced apart from each other, the length of 10 to 2000 ㎛, 1 to 20 ㎛ in height.

In the present invention, the attached neurons are characterized in that the average density is maintained at 10 to 300 cell / mm2.

More specifically, the average density of neurons attached to the neural cell adhesion layer provided on the first channel, the second channel, and the base surface of the microfluidic channel is maintained at 10 to 300 cell / mm 2, and the cell survival rate is higher than a certain level. Can be maintained.

In addition, the adhesion and growth of the injected nerve cells in each flow path is progressed, each flow path is hydrodynamically connected to equalize the culture conditions through the exchange of fluid, individual neurons or a few nerve cells in a specific flow path (1 cell / mm 2) has the advantage of being able to grow at a very high survival rate.

According to the present invention, after culturing attached neurons after injection at different densities in each flow path separated from each other, cultured neurons having high density and low density are moved in one direction by a concentration gradient through a microfluidic channel. It provides a method for forming an artificial synapse having a direction comprising a step of synapses formed between dendritic projections or cell bodies of neurons grown at low density.

More specifically, neurons grown under normal culture conditions form synapses in a disordered direction, and it is very difficult to distinguish each of the protrusions located around one neuron. However, it is very easy to distinguish between cell bodies, axons and dendrites in individual neurons cultured in spatial isolation.

Synapse formation method of the present invention has the advantage of forming a synapse having a direction between the isolated axons and the spatially separated axons using the surface engineering and MEMS (Micros Electro Mechanical System) process under normal neuron culture conditions have.

The method for separating axons according to the present invention can maintain the average density of neurons at 10 to 300 cell / mm 2 by using the microfluidic chip for separating the axons. Neurons (1 cell / mm ² ) have the advantage of maintaining a very high survival rate.

The method for separating axons according to the present invention uses the microfluidic chip for separating axons, thereby allowing the cell bodies of the neurons in the low density compartment to recognize signals formed from environmental changes around the cell bodies of the neurons in the high density compartment. Will greatly contribute to the manufacture of biochips.

1 is a conceptual diagram of a microfluidic chip according to the present invention,
(A: polymer microfluidic chip irreversibly attached to the substrate, B: polymer microfluidic chip designed to be reversibly separated on the substrate)
2 is a conceptual diagram for synapse formation having a direction of the neurons using the microfluidic chip according to the present invention,
3 is a result of confirming that the axon projections are spatially separated from the cell body and the dendrite using a microfluidic chip coated with a neuronal cell adhesion material according to the present invention,
(A: conceptual diagram, B: micrograph, C: immunochemical analysis)
4 is a result of confirming that the axon projections are spatially separated from the cell body and the dendrite using a microfluidic chip formed with a pattern of neuronal cell adhesion material according to the present invention.
(A: conceptual diagram, B: micrograph, C: immunochemical analysis)
5 is a result of confirming that the axon projections are spatially separated from the cell body and the dendrites by using the microfluidic chip in which the polymer casting microstructures according to the present invention are formed.
(A: conceptual diagram, B: micrograph, C: immunochemical analysis)
6 is a result of confirming that the axon projections are spatially separated from the cell body and the dendrite using a microfluidic chip in which the photosensitive polymer microstructure according to the present invention is formed.
(A: conceptual diagram, B: micrograph, C: immunochemical analysis)
Figure 7 shows the survival rate of neurons cultured using the microfluidic chip according to the present invention,
(A: culture photo, B: enlarged photo)
Figure 8 shows the growth of neurons cultured using the microfluidic chip according to the present invention over time,
9 is a photo showing the result of forming a snap around the cultured nerve cells using the microfluidic chip according to the present invention,
10 shows cell bodies, dendrites, and axons formed from neurons cultured using the microfluidic chip according to the present invention, respectively.
(A: micrograph, B: immunochemical analysis)
Figure 11 shows a picture of the synapse formed around the nerve cells cultured using the microfluidic chip according to the present invention.
(A: micrograph, B: immunochemical analysis)

Hereinafter, a method of manufacturing a gap device according to the present invention will be described with reference to the following examples, but the examples presented do not limit the scope of the present invention.

Example 1 Isolation of Spatial Axons from Neurons

(1) Separation of axons from spatial differences

It has a large number of compartments by irreversibly attaching a polymer chip having compartments (100 μm in height, 800 to 1000 μm in width) connected to the substrate by microfluidic channels (height 3 to 5 μm, width 50 μm, length 100 to 1000 μm). A microfluidic chip was produced. After applying poly-L-lysine (PLL) to the substrate, high density neurons (300 cell / mm 2) were injected into one compartment of the microfluidic chip.

As can be seen in Figure 3, as the nerve cells grow, it was confirmed that the axon projections of the nerve cells grown at the attachment portion grew to empty spaces ((B) of FIG. 3) when stained by immunochemical methods, the blanks It can be seen that the projections listed in space are axon projections (FIG. 3C).

(2) Separation of axons using surface patterning

Poly-L-lysine (PLL) -coated substrate using a polymer coating and air plasma pattern of poly-L-lysine (PLL) 5 ㎛, interval 5 ㎛ pattern 800 ㎛, interval 800 After forming to form a pattern by performing to the μm, neurons were selectively attached using a microfluidic chip having the same compartment as in Example 1.

As can be seen in Figure 4, as the nerve cells grow, the axon projections of the nerve cells grown in the attachment part were confirmed to grow in the pattern part (Fig. 4 (B)), when stained by the immunochemical method, the pattern It can be confirmed that the protrusions grown into the space are axon protrusions (FIG. 4C).

(3) Separation of axons using polymer casting microstructures

Polymer casting microstructures were fabricated using prefabricated formwork using semiconductor process technology. The height of the microstructure is 3 to 5 ㎛, the width of the microstructure is 50 ㎛, the pattern for cell adhesion between the microstructure and the microstructure is 10 ㎛.

Neurons were selectively attached to the polymer substrate coated with poly-L-lysine (PLL) using a microfluidic chip having compartments.

As can be seen in Figure 5, as the nerve cells grow, the axon projections of the nerve cells grown in the attachment portion grew into a pattern portion between the microstructures and the microstructures (Fig. 5 (B)), stained by immunochemical methods Even if it was confirmed that the projections grown in the pattern space is axon projection (Fig. 5 (C)).

(4) Separation of Axons Using Photosensitive Polymer Microstructures

After the photosensitive polymer (SU8 5) was coated on the glass substrate to a thickness of 3 to 5 μm, the photosensitive polymer microstructure was fabricated using a photomask prepared by printing on a film. 1 (b)) the height of the microstructure is 3 ~ 5㎛ the width of the obstacle is 50㎛, the pattern for cell adhesion between the microstructure and the microstructure is 10㎛.

Poly-L-lysine (PLL) was applied to the glass substrate on which the photosensitive polymer microstructure was patterned, and then neurons were selectively attached using a microfluidic chip having a compartment.

As can be seen in Figure 6, as the nerve cells grow, the axon projections of the nerve cells grown at the attachment part grew into the pattern part (Fig. 6 (B)), even if the pattern is divided by the immunochemical method space Growing protrusions could be confirmed that the axon protrusion (Fig. 6 (C)).

[ Example  2] Investigation of growth of axons of neurons over time

The microfluidic chip having the compartments (100 μm in height and 800 to 1000 μm in width) connected to the substrate by microfluidic channels (3 to 5 μm in height, 50 μm in width and 100 to 1000 μm in length) is irreversibly attached to many substrates. A microfluidic chip having was prepared. After applying poly-L-lysine (PLL) to the substrate, one channel (compartment) of the microfluidic chip is injected with high density nerve cells (300 cell / mm 2), and the adjacent channel (Compartments) were grown by injecting neurons (1 cell / mm 2) of low density.

As can be seen in Figure 8 neurons injected and grown in each passage (compartment) as a result of confirming the growth process of the central nervous system neurons with the passage of time, after 7 hours to reach the first stage (lamellipodia), After 18 hours, one can observe the second step, the minor processes. In addition, when 28 hours have elapsed, a third step, the adriatic outgrowth stage, was reached, and the process continued up to 40 hours.

These results confirm that neurons cultured in the microfluidic chip faithfully follow the neuronal growth stage suggested by CG Dotti et al. (J. Neurosci. 1988, 8, 1454-1468.). .

[ Example  3] formation of directional synapses

In FIG. 1, a microfluidic chip according to the present invention comprises a section (100 μm in height and 800 to 1000 μm in width) connected to a substrate by a microfluidic channel (height 3 to 5 μm, width 50 μm, length 100 to 1000 μm). Irreversibly attach the microfluidic chip having a microfluidic chip (3-5 μm in height, 50 μm in width, 100-1000 μm in length) using a photosensitive polymer to the microfluidic chip and glass substrate having many compartments, A microfluidic chip having many compartments was prepared by reversibly attaching a microfluidic chip having a large number of compartments (height 100 μm and a width of 800 to 1000 μm).

After applying poly-L-lysine (PLL) to the substrate, one channel (compartment) of the microfluidic chip is injected with high density nerve cells (300 cell / mm 2), and the adjacent channel (Compartments) were grown by injecting neurons (1 cell / mm 2) of low density.

As shown in FIG. 9, 5 days after the injection of neurons, the axons and dendrites were separated from individual neurons so as to be distinguishable from the cell body in the left compartment, and the axons were separated from the right compartment to the left through the microfluidic channel. It was confirmed that a synapse having a directional growth was formed in the compartment.

That is, in FIG. 10, the axons separated in the flow path (compartment) of the microfluidic chip grown at high density form a synapse with the dendritic projections or cell bodies of the neurons grown at low density in the neighboring flow path (compartment). there was. When distinguished and stained by immunochemical methods, axons, dendrites, and cell bodies could be distinguished, and the synapses formed between the axons and dendrites or cell bodies separated in FIG. 11 could be identified.

In the present invention as described above has been described by the specific material or specific matters and the embodiments and drawings, which are provided only to help a more general understanding of the present invention, the present invention is not limited to the above embodiments, Various modifications and variations can be made by those skilled in the art to which the present invention pertains.

Therefore, the spirit of the present invention should not be limited to the described embodiments, and all of the equivalents or equivalents of the claims as well as the claims to be described later will belong to the scope of the present invention. .

Claims (11)

A first flow passage having a first inlet and a first outlet, a second flow passage having a second inlet and a second outlet, a microfluidic channel connecting the first and second flow passages, the first flow passage, and a second flow passage In the axon projection separation method using a microfluidic chip for axon projection separation comprising a nerve cell adhesion layer provided on the base surface of the microfluidic channel,
Selectively injecting a first suspension containing neurons into the first inlet and attaching to the neuronal cell adhesion layer; And
Separating and growing axons in the microfluidic channel through the culture of the attached nerve cells;
A method of separating the spatial axon projections from nerve cells using a microfluidic chip for axon separation. The method of claim 1,
The separation method of the axon projection is characterized in that the second suspension is injected through the second inlet is a suspension containing the nerve cells of a lower density than the first suspension. 3. The method according to claim 1 or 2,
The nerve cell attached to the nerve cell adhesion layer of the first flow path or the second flow path is separated from the axon projections, characterized in that the movement is separated by a concentration gradient through the microfluidic channel in one direction of high density to low density. The method of claim 1,
The microfluidic chip is made of at least one polymer or photosensitive polymer selected from polydimethylsiloxane (PDMS), polyurethane (PU), and polymethyl methacrylate (PMMA) on a substrate. Separating the axon projections, characterized in that to form a flow path and a microfluidic channel by attaching a microstructure. The method of claim 1,
The flow path has a section of 100 to 1000 ㎛, 1 to 20 mm in length, 50 to 500 ㎛ in height, connected to the microfluidic channel, characterized in that the separation of the axon projections. 6. The method of claim 5,
The microfluidic channel is a separation method of the axon projections, characterized in that a plurality of microfluidic channels separated from each other of 3 to 100 ㎛ in width, 10 to 2000 ㎛ in length, 1 to 20 ㎛ in height. The method of claim 1,
The neuron adhesion layer is formed on at least one substrate selected from glass, silicon, silicon oxide, silicon nitride, polycarbonate, polystyrene, and polypropylene. Separation method of the axon projections, characterized in that located. The method of claim 7, wherein
The neuronal cell adhesion layer is a poly-L-lysine (PLL), poly-D-lysine (poly-D-lysine, PDL), laminin (laminin), laminin (laminin) structure A method for separating axons, characterized in that it comprises one or more neuronal cell attachment substances selected from polypeptides or oligopeptides. The method of claim 1,
The attached neurons are separated from the axon projections, characterized in that the average density is maintained at 10 to 300 cell / ㎜. Axon projections of rat neurons isolated by the method of any one of claims 1 or 2 according to the axon separation. A method for forming an artificial synapse having directivity, comprising the step of synaptic formation between dendrites or cell bodies of neurons grown at low density and axons according to claim 10.

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