U.S. patent number 10,385,306 [Application Number 15/536,748] was granted by the patent office on 2019-08-20 for device and method for single cell screening based on inter-cellular communication.
This patent grant is currently assigned to UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY). The grantee listed for this patent is UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY). Invention is credited to Cedric Bathany, Yoon Kyoung Cho, Devrim Gozuacik, Jun Young Kim.
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United States Patent |
10,385,306 |
Cho , et al. |
August 20, 2019 |
Device and method for single cell screening based on inter-cellular
communication
Abstract
A device for single-cell analysis according to an embodiment of
the present invention comprises: a substrate; a gap between the
substrate and porous membrane which is a space for culture medium;
and a porous membrane formed on having a pore capable of isolating
a second cell into single cell units. A method for single-cell
analysis according to an embodiment of the present invention
comprises: Culturing a first cell in a culture medium on a bottom
side of porous membrane; Applying a sample including a second cell
on a porous membrane in a culture medium; Isolating the second cell
into single cell units in a pore existing in the porous membrane
with a external force such as agitation and gravitational force;
Generating an interaction situation between the first cells and the
single cell-level second cell; Analyzing a cellular phenomena of
the first cell or the second cell.
Inventors: |
Cho; Yoon Kyoung (Ulsan,
KR), Bathany; Cedric (Ulsan, KR), Kim; Jun
Young (Ulsan, KR), Gozuacik; Devrim (Istanbul,
TR) |
Applicant: |
Name |
City |
State |
Country |
Type |
UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) |
Ulsan |
N/A |
KR |
|
|
Assignee: |
UNIST(ULSAN NATIONAL INSTITUTE OF
SCIENCE AND TECHNOLOGY) (Ulsan, KR)
|
Family
ID: |
56126993 |
Appl.
No.: |
15/536,748 |
Filed: |
December 18, 2015 |
PCT
Filed: |
December 18, 2015 |
PCT No.: |
PCT/KR2015/013959 |
371(c)(1),(2),(4) Date: |
June 16, 2017 |
PCT
Pub. No.: |
WO2016/099207 |
PCT
Pub. Date: |
June 23, 2016 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180002654 A1 |
Jan 4, 2018 |
|
Foreign Application Priority Data
|
|
|
|
|
Dec 18, 2014 [KR] |
|
|
10-2014-0183605 |
Mar 11, 2015 [KR] |
|
|
10-2015-0033711 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M
1/34 (20130101); G01N 33/5005 (20130101); C12M
25/02 (20130101); G01N 33/48 (20130101); C12M
41/46 (20130101); C12M 23/16 (20130101); G01N
33/54366 (20130101) |
Current International
Class: |
C12M
1/34 (20060101); C12M 3/06 (20060101); C12M
1/12 (20060101); G01N 33/48 (20060101); G01N
33/543 (20060101); G01N 33/50 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
10-2011-0035213 |
|
Apr 2011 |
|
KR |
|
10-2011-0068170 |
|
Jun 2011 |
|
KR |
|
10-2014-0106933 |
|
Sep 2014 |
|
KR |
|
10-1458425 |
|
Nov 2014 |
|
KR |
|
Other References
Danilevicius et al. J of Biomedical Optics, 2012, 17(8):1-7. cited
by examiner .
Fillinger et al. J of Vascular Surgery, 1993, 17:1058-1068. cited
by examiner .
Yamaguchi et al. Sensors and Actuators B, 2009, 136:555-561. cited
by examiner.
|
Primary Examiner: Shen; Bin
Attorney, Agent or Firm: Lex IP Meister, PLLC
Claims
What is claimed is:
1. A device for single-cell analysis comprising: a substrate; a
culture medium disposed on the substrate, the culture medium
includes a plurality of first cells cultured therein; a porous
membrane disposed on the culture medium, the porous membrane has a
plurality of pores capable of isolating a cell into single cell
units; and a plurality of second cells isolated in some of the
pores of the porous membrane, wherein a gap is formed between the
substrate and the porous membrane which is a space for the culture
medium, wherein the gap continues throughout below the porous
membrane, wherein each of the pores penetrates the porous membrane
from top surface to bottom surface, wherein a diameter of the pore
is 1 to 100 .mu.m, and wherein the first cell is a fibroblast cell
and the second cell is a tumor cell.
2. The device of claim 1, wherein the gap between the porous
membrane and the substrate is 1 to 100 .mu.m.
3. The device of claim 1, wherein the porous membrane is made of a
material selected from polymeric or inorganic materials.
4. The device of claim 3, wherein the porous membrane is made of a
photosensitive polymeric material.
5. The device of claim 4, wherein the porous membrane is made by
forming a pore in a photosensitive polymeric membrane through a
lithography method.
6. The device of claim 3, wherein the porous membrane is made by
forming a pore in a polymeric membrane through a soft lithography
method.
7. The device of claim 1, wherein the porous membrane has pores of
10.sup.2 to 10.sup.6 holes/cm.sup.2.
8. The device of claim 1, wherein the porous membrane has pores and
an interval between the pores is 1 .mu.m to 10 mm.
9. A method for single-cell analysis using the device of claim 1
comprising: Culturing a first cell in a culture medium on a bottom
side of porous membrane; Applying a sample including a second cell
on a porous membrane in a culture medium; Isolating the second cell
into single cell units in a pore existing in the porous membrane
with an external force such as agitation and gravitational force;
Generating an interaction situation between the first cells and the
single cell-level second cell; Analyzing cellular phenomena of the
first cell or the second cell.
10. The method of claim 9, wherein the first cell is a fibroblast
cell and the second cell is a tumor cell.
11. The method of claim 9, wherein a thickness of the culture
medium is 1 to 100 .mu.m.
12. The method of claim 9, wherein a concentration of the first
cell is 1.times.10.sup.5 to 1.times.10.sup.7 cells/mL.
13. The method of claim 9, wherein when applying the second cells,
stirring is performed at the same time.
14. The method of claim 13, wherein the stirring is performed for 1
minute to 1 hour at 10 to 500 rpm.
15. The method of claim 9, wherein a concentration of the second
cell in the sample i (a number of pores in a porous
membrane.times.1) to (a number of pores in a porous
membrane.times.10,000) cells/mL or 1.times.10.sup.2 to
1.times.10.sup.10 cells/mL.
16. The method of claim 9, wherein a diameter of the pore is 1 to
100 .mu.m.
17. The method of claim 9, wherein the porous membrane has pores of
10.sup.2 to 10.sup.6 holes/cm.sup.2.
18. The method of claim 9, wherein the porous membrane has pores
and a gap between the pores is 1 .mu.m to 10 mm.
19. The method of claim 9, wherein the interaction is generated by
contact or paracrine factors between the first cell and the second
cell for 1 hour to 7 days.
20. The method of claim 9, wherein the analyzing a cell activity of
the first cell or the second cell further comprises screening the
cell activity of the first cell or the second cell.
21. The method of claim 9, wherein the analyzing a cell activity of
the first cell or the second cell further comprises capturing and
analyzing the second cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
This application is a National Stage Application of PCT Application
PCT/KR2015/013959, filed on Jun. 26, 2017, which claims priority of
Korean Patent Application No. 10-2014-018365 filed on Dec. 18, 2014
and Korean Patent Application No. 10-2015-0033711 filed on Mar. 11,
2015, the contents of which are incorporated herein by
reference.
FIELD OF THE INVENTION
The present invention relates to a device and a method for single
cell level screening based on interaction among single cell and
neighboring multiple cells.
DESCRIPTION OF THE RELATED ART
In cellular microenvironment, cells give and receive messages with
its environment and with itself via cytokine signals and/or direct
contact affecting cellular phenotypes. For this significance, in
vitro platform for cell-to-cell interaction was actively developed
in a form of 2-D or 3-D platform to mimic and to investigate the
interactions between cell populations. But it can obtain only
average results from many numbers of cells. This in turn motivates
the development of complementary in vitro platform of single cell
isolation and its analysis.
Single cell isolation techniques have been developed by using
microwell arrays, traps using hydrodynamic fluid control,
dielectrophoresis and surface micropatterning etc. Using these
single cell isolation techniques, they used these single cell
isolation techniques in various application such as analysis of
heterogeneous cellular phenotype, paracrine factor secretion and
DNA repair capacities with different genetic backgrounds.
Specifically, single cell pairing techniques have been highlighted
because it can achieve not only the spatiotemporal control of
cellular interaction but also make a special situation for single
cell level interaction. The application includes cell migration,
proliferation patterns of stem cell, and heterogeneous dynamics of
CD8 T cells through interaction with lymphocyte. It can provide a
single cell level resolution in resolving stochastic cellular
behavior in large populations, which helps to understand the cell
dynamics and to achieve better statistical data of intercellular
signaling mechanisms unlike conventional bulk system. The
previously reported single cell pairing method has a limit which
focuses on only single cell and single cell interaction. There is a
gap between the in-vitro single cell and single cell interaction
chips and in-vivo cellular microenvironment. For example, tumor
cells are situated in a microenvironment surrounded by multiple
stromal cells and interact each other.
CONTENTS OF THE INVENTION
Problem to be Solved
The purpose of the present invention is to provide a device and a
method for screening cells in a single cell level based upon
intercellular communication between single cell and neighboring
multiple cells.
Means for Solving Problem
A device for single-cell analysis according to an embodiment of the
present invention comprises: a substrate; a gap between membrane
and substrate and capable of culturing a first cell; and a porous
membrane having a pore capable of isolating a second cell into
single cell units.
A gap between the porous membrane and the substrate may be 1 to 100
.mu.m.
The porous membrane may be selected from polymeric or inorganic
materials.
The porous membrane may be made by forming a pore in a polymeric
membrane through a soft lithography method.
The porous membrane may be a photosensitive polymeric material.
The porous membrane may be made by forming a pore in a
photosensitive polymeric membrane through a lithography method.
A diameter of the pore may be 1 to 100 .mu.m.
The porous membrane may have pores of 10.sup.2 to 10.sup.6
holes/cm.sup.2.
The porous membrane may have pores and a gap between the pores may
be 1 .mu.m to 10 mm.
A method for single-cell analysis according to an embodiment of the
present invention comprises: Culturing a first cell in a culture
medium on a bottom side of porous membrane; Applying a sample
including a second cell on a porous membrane in a culture medium;
Isolating the second cell into single cell units in a pore existing
in the porous membrane with a external force such as agitation and
gravitational force; Generating an interaction situation between
the first cells and the single cell-level second cell; Analyzing a
cellular phenomena of the first cell or the second cell.
The first cell may be a fibroblast cell and the second cell may be
a tumor cell.
A gap between porous membrane and substrate may be 1 to 100
.mu.m.
A concentration of the first cell may be 1.times.10.sup.5 to
1.times.10.sup.7 cells/mL.
A concentration of the second cell in the sample may be (a number
of pores in a porous membrane.times.1) to (a number of pores in a
porous membrane.times.10,000) cells/mL or 1.times.10.sup.2 to
1.times.10.sup.10 cells/mL.
When applying the external force such as gravitation force,
stirring may be performed at the same time. Moreover, the stirring
may be performed for 1 minute to 1 hour at 0 to 500 rpm.
A diameter of the pore may be 1 to 100 .mu.m.
The porous membrane may have pores of 10.sup.2 to 10.sup.6
holes/cm.sup.2.
The porous membrane may have pores and a gap between the pores may
be 1 .mu.m to 10 mm.
The interaction may be generated by contact and paracrine
communication between the first cells and the second cell for 1
hour to 7 days.
The analyzing of cellular activities of the first cell or the
second cell may further comprise monitoring the cellular activities
of the first cells or the second cell.
The analyzing of cellular activities of the first cells or the
second cell may further comprise obtaining and analyzing the first
cells.
The analyzing of cellular activities of the first cells or the
second cell may further comprise capturing and analyzing the second
cell.
Effects of the Invention
A cellular phenomenon monitoring and analysis at single cell units
according to the single cell-to-bulk cells interaction may be
easily performed. A gene analysis can be available as well as a
visual analysis at single cell units.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic view illustrating a device for single-cell
analysis according to an embodiment of the present invention.
FIG. 2 is a close-up photograph of a porous membrane with
pores.
FIG. 3 is a schematic view illustrating a device for single-cell
analysis when culturing a first cell on bottom side of a porous
membrane.
FIG. 4 is a schematic view illustrating a device for single-cell
analysis when isolating a second cell into a pore of a porous
membrane.
FIG. 5 is a photograph of a device for single-cell analysis
according to an embodiment of the present invention.
FIG. 6 is a flowchart of a method for single-cell analysis
according to an embodiment of the present invention.
FIG. 7 is a close-up photograph of a tumor cell isolated in a
pore.
FIG. 8 is a close-up photograph of screening a fibroblast existing
on a bottom side of porous membrane and generating an autophagy
phenomenon by an interaction with an isolated single tumor
cell.
FIG. 9 is a graph to explain a monitor performance of the present
invention, which holes with single cell have a significant
difference between empty hole in case of an percentage of autophagy
phenomenon in fibroblasts.
FIG. 10 is a photograph of isolation of a single tumor cell from a
pore and genomic result using this single tumor cell.
DETAILED DESCRIPTION
The terminology used in the specification is for the purpose of
referring to particular embodiments by way of example only. Thus,
the terminology is not intended to be limiting of the present
invention. The singular forms used in the specification include the
plural forms, unless the context clearly dictates otherwise. The
term "comprising" used in the specification specify the specific
characteristics, regions, integers, steps, operations, elements,
and/or components, but do not preclude the presence or the addition
of other specific characteristics, regions, integers, steps,
operations, elements, and/or components.
Unless otherwise defined, all terms including technical and
scientific terms used herein have the same meaning as commonly
understood by those skilled in the art of the present invention.
The terms defined in commonly used dictionaries are additionally
interpreted as having a meaning that is consistent with the
relevant technical literature and current disclosure, but are not
interpreted in an idealized or overly formal sense unless expressly
defined herein.
Hereinafter, embodiments of the present invention will be described
in detail. However, it is for illustrative purpose only and not
meant to limit or otherwise narrow the scope of the present
invention. Therefore, the present invention will only be defined by
the appended claims.
FIG. 1 schematically shows a device for single-cell analysis
according to an embodiment of the present invention. The device for
single-cell analysis in FIG. 1 is intended to be merely
illustrative of the present invention, and the present invention is
not limited thereto. Thus, a device for single-cell analysis may be
modified in various ways.
As shown in FIG. 1, a device for single-cell analysis according to
an embodiment of the present invention comprises: a substrate; a
gap between the substrate and porous membrane which is a space for
culture medium; and a porous membrane formed on having a pore
capable of isolating a second cell into single cell units. The
second cell (200) isolated in the pore (31) as a single cell
performs an interaction with the first cell (100) cultured in the
culture medium, and then the second cell (200) together with the
porous membrane (30) is separated from the first cell (100). Thus,
the second cell (200) may be analyzed as single cell units. As a
result, an analysis of single cell units according to a single
cell-to-bulk cells interaction may be easily performed. Moreover, a
gene analysis can be available as well as a visual analysis at
single cell units.
A gap between the porous membrane (30) and the substrate may be 1
to 100 .mu.m. If the gap between the porous membrane (30) and the
substrate is too narrow, a culture of the first cell (100) on the
bottom side of porous membrane is difficult. If the gap between the
porous membrane (30) and the substrate is too big, the second cell
(200) is not isolated in the pore (31), but passed through the gap
between the porous membrane (30) and the substrate. In particular,
the gap between the porous membrane (30) and the substrate may be 1
to 100 .mu.m.
The porous membrane (30) may be a polymeric material. More
specifically, the polymeric material are polymethyl(meth)acrylate
(PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC),
polyethylene terephthalate (PET), polypropylene (PP), and the like.
When using a polymeric material as the porous membrane (30), a soft
lithography method may be used to form the pore (31) in the porous
membrane (30). When using a photosensitive polymeric material as
the porous membrane (30), a lithography method may be used to form
the pore (31) in the porous membrane (30). Describes an example of
the process of forming the pore (31) in the porous membrane (30)
through the soft lithography method is as follows. Deposit the
photoresist on silicon wafer. Make a pattern using
photolithography, Add pre-cured PDMS and cured, Peel away PDMS
master, Do RIE treatment with CHF3, Inject pre-cured PDMS into the
gap between PDMS master and glass, Release PDMS membrane where the
pore (31) is formed. A close-up photograph of the porous membrane
(30) with a pore (31) is described in FIG. 2.
Slide glass or polydimethylsiloxane (PDMS) may be used as the
substrate. A device for single-cell analysis may be obtained by
using a soft etching method (soft lithography) on the substrate to
secure a space, by processing a surface of the substrate. The
method stated above is only one example to prepare the device for
single-cell analysis and may vary depending on needs.
A diameter of the pore (31) formed in the porous membrane (30) may
be 1 to 100 .mu.m. If the diameter of the pore (31) is too small,
the second cell (200) is difficult to be isolated in the pore (31).
If the diameter of the pore (31) is too big, the second cell (200)
may be not isolated as single cell units.
The porous membrane (30) may have pores of 10.sup.2 to 10.sup.6
holes/cm.sup.2. If the pore (31) is too small, the amount of the
second cell (200) for an analysis may become too small. If the pore
(31) is too large, there is a problem that a device for analysis of
the second cell (200) may become large.
A gap between the pores (31) formed in the porous membrane (30) may
be 1 .mu.m to 10 mm. If the gap between the pores (31) is too
narrow, an interaction between the neighboring first cells (100)
cultured in the culture medium occurs. Thus, an analysis of a
cellular phenomenon caused by an interaction between the first cell
(100) and the second cell (200) may be difficult. If the gap
between the pores (31) is too wide, there is a problem that a
device for analysis of the second cell (200) may become large.
A device for single-cell analysis according to an embodiment of the
present invention may further comprise a reservoir, porous
membrane, gap between porous membrane and substrate.
FIG. 3 schematically describes a culture of the first cell (100) on
the bottom side of the membrane (20).
FIG. 4 schematically describes an isolation of the second cell
(200) in the pore (31) of the porous membrane (30). By applying the
external forces such as agitation and gravitational force, the
second cell (200) is isolated in the pore (31) at single cell
units.
FIG. 5 describes a photograph of a device for single-cell analysis
according to an embodiment of the present invention.
FIG. 6 schematically describes a flowchart of a method for
single-cell analysis according to an embodiment of the present
invention. The flowchart of a method for single-cell analysis in
FIG. 6 is intended to be merely illustrative of the present
invention, and the present invention is not limited thereto. Thus,
a method for single-cell analysis may be modified in various
ways.
As shown in FIG. 6, a method for single-cell analysis comprises:
culturing a first cell (100) on the bottom side of porous membrane
(20) formed on the gap between a porous membrane and a substrate
(S10): applying a sample including a second cell (200) on a porous
membrane (30) in the culture medium (20) (S20); isolating the
second cell (200) into single cell units in a pore (31) existing in
the porous membrane (30) with a external force such as agitation
and gravitational force (20) (S30); generating an interaction
situation between the first cells (100) and the single cell-level
second cell (200) (S40);and analyzing a cellular activity of the
first cells (100) or the second cell (200).
First, in step S10, the first cell (100) is cultured on the bottom
side of a porous membrane. A thickness of a gap between the
substrate and the porous membrane may be 1 to 100 .mu.m. If the gap
between the porous membrane (30) and the substrate is too narrow, a
culture of the first cell (100) in the culture medium is difficult.
If the gap between the porous membrane (30) and the substrate is
too wide, the second cell (200) is not isolated in the pore (31),
but passed through the culture medium.
When culturing the first cell (100) on the bottom side of the
porous membrane, the medium containing first cell (100) is put on
the bottom side of the porous membrane for few hours (20) and the
porous membrane is attached with substrate, which gap between the
porous membrane and substrate can supply the nutrient. A
concentration of the first cell (100) may be 1.times.10.sup.5 to
1.times.10.sup.7 cells/mL.
FIG. 3 schematically describes a culture of the first cell (100) on
the bottom side of the porous membrane (20). The medium containing
first cell (100) is put on the bottom side of the porous membrane
for few hours (20) and the porous membrane is attached with
substrate
If a cell can induce an interaction with the second cell (200), the
cell may be used as the first cell (100) without restriction. In
particular, the first cell may be a fibroblast cell.
In step S20, a sample including the second cell (200) is applied on
the porous membrane (30) with external forces such as agitation and
gravitational forces. The sample includes the second cell (200) as
well as a medium, wherein a concentration of the second cell (200)
in the sample may be (a number of pores in a porous
membrane.times.1) to (a number of pores in a porous
membrane.times.10,000) cells/mL or 1.times.10.sup.2 to
1.times.10.sup.10 cells/mL. If the concentration of the second cell
(200) is too low, the efficiency of the analysis may be reduced
because empty pores (31) in which the second cell (200) is not
isolated become a lot. If the concentration of the second cell
(200) is too high, the second cell is difficult to be isolated in
the pore (31) as single cell units.
If a cell can induce an interaction with the first cell (200) to
analyze changes of cellular activities after the interaction, the
cell may be used as the second cell (200) without restriction. In
particular, the second cell may be a tumor cell.
In step S30, the second cell (200) is isolated into single cell
units in a pore (31) existing in the porous membrane (30) by
applying external forces such as agitation and gravitational
forces.
FIG. 4 schematically describes an isolation of the second cell
(200) in the pore (31) of the porous membrane (30). By applying
external forces such as agitation and gravitational forces in the
direction of the arrow, the second cell (200) is isolated in the
pore (31) at single cell units.
When applying external forces such as agitation and gravitational
forces, a sample including the second cell (200) in the reservoir
is directed into the pore (31) formed on the porous membrane (30).
Untrapped second cells are washed and then, only the trapped second
cell (200) is isolated in the pore (31) in a single-cell state. At
this time, the applied agitation velocity may be 0 to 200 rpm. For
example, the stirring may be performed by a method of putting the
device for single-cell analysis on a shaker. The stirring may be
performed for 1 minute to 1 hour at 10 to 500 rpm. If the agitation
velocity is too slow, the second cells are hard to spread. If the
agitation velocity is too fast, the second cells (200) tend to
gather on edge part.
When applying the agitation force, different number of second cells
input may be performed at the same time. The number of second cells
input may be varied from 1*number of total pores to 1,000*number of
total pores. If a input number of second cells is too low, there
may be a problem in efficiency of single cell entrapment is low. If
a input number of second cells is too many, a percentage of
multiple cell entrapment is increased Thus, there is a problem that
the cell is isolated only to a specific part in single cell
level.
A diameter of the pore (31) isolating the second cell (200) may be
1 to 100 .mu.m. If the diameter of the pore (31) is too small, the
second cell (200) is difficult to be isolated in the pore (31). If
the diameter of the pore (31) is too wide, the second cell (200)
may be not isolated as single cell units.
The porous membrane (30) may have pores of 10.sup.2 to 10.sup.6
holes/cm.sup.2. If the pore (31) is too small, there is a problem
that the amount of the second cell (200) for an analysis may become
too small. If the pore (31) is too large, the efficiency of the
analysis may be reduced because empty pores (31) in which the
second cell (200) is not isolated become a lot.
A gap between the pores (31) isolating the second cell (200) may be
1 .mu.m to 10 mm. If the gap between the pores (31) is too narrow,
an interaction between the neighboring first cells (100) cultured
on the bottom side of porous membrane occurs. Thus, an analysis of
a cellular phenomenon caused by an interaction between the first
cell (100) and the second cell (200) may be difficult. If the gap
between the pores (31) is too wide, there is a problem that a
device for analysis of the second cell (200) may become large.
In step S40, an interaction is generated by contact or paracrine
factor between the first cell (100) and the second cell (200). At
this time, the interaction is generated for 1 hour to 7 days. For
example, the interaction may be caused by directly contacting
between a tumor cell and a fibroblast cell or by an indirect
paracrine factor.
A method of analyzing may be screening of the cell activity changed
by the interaction between the first cells (100) or the second
cells (200), or capturing and analyzing the second cell (200)
completing the interaction. The second cell (200) completing the
interaction exists inside of the pore (31) of the porous membrane
(30) in an isolated state, so single cell units of the second cell
(200) may be analyzed by separating the second cell (200) form the
first cell (100).
In particular, the interaction between the first cell (100) and the
second cell (200) may be analyzed by a green marker previously
inserted in the first cell (100). Moreover, a gene analysis may be
performed by obtaining the second cell (200) as single cell units
through a single cell picker (Kuiqpick) and analyzing the obtained
second cell (200) through a single cell genetic analysis device
(Biomark HD).
Therefore, the visual analysis as well as the gene analysis of
single cell units can be available.
Below a preferred embodiment of the present invention and
comparative examples will be described. However, embodiment stated
below is just an embodiment of the present invention, so the
present invention is not limited thereto.
EXAMPLE 1
A porous membrane having 5,000 pores whose pore size is 30 .mu.m
was prepared by using a Polydimethylsiloxane (PDMS). A
Polydimethylsiloxane (PDMS) coated substrate was prepared as a
substrate having 5 .mu.m thickness. The substrate was used as a
space of culture medium. FIG. 7 is a close-up photograph of a tumor
cell isolated in a pore. A tumor cell was isolated in the pore as
single cell units by applying a sample including a tumor cell on
the porous membrane, by applying 10,000 input number of second
cells, and by stirring for 5 minutes at 100 rpm.
Table 1 shows yield efficiency obtained by organizing a number
ratio of the tumor cell isolated in the pore against a number of
the tumor cell applied on the porous membrane.
FIG. 8 is a close-up photograph of screening a fibroblast existing
on a bottom side of porous membrane and generating an autophagy
phenomenon by an interaction with an isolated single tumor
cell.
We observed whether a cell change of a fibroblast cell occurs by
performing interaction between a tumor cell and a fibroblast cell
for 6 hours. Thus, we can found that there was an interaction with
the second cell isolated in the pore and the first cell.
FIG. 9 is a graph to explain a monitor performance of the present
invention, which holes with single cell have a significant
difference between empty hole in case of an percentage of autophagy
phenomenon in fibroblasts.
Table 2 shows comparison between empty holes and holes with single
tumor in case of autophagy activation percentage in
fibroblasts.
FIG. 10 is a photograph of isolation of a single tumor cell from a
pore and genomic result using this single tumor cell. We observed
that the proteins extracted from isolated single cell can be used
to do gene analysis.
EXAMPLE 2
The stirring speed was adjusted to 0 rpm. The rest of the
experiments were performed in the same manner as in Example 1.
EXAMPLE 3
The stirring speed was adjusted to 200 rpm. The rest of the
experiments were performed in the same manner as in Example 1.
EXAMPLE 4
The number of second cells input was adjusted to 5,000. The rest of
the experiments were performed in the same manner as in Example
1.
EXAMPLE 5
The number of second cells input was adjusted to 20,000. The rest
of the experiments were performed in the same manner as in Example
1.
TABLE-US-00001 TABLE A Number of Stirring Stirring Yield second
cells speed time efficiency input (rpm) (min) (%) Example 1 10,000
100 5 ~50 Example 2 10,000 0 5 ~40 Example 3 10,000 200 10 ~35
Example 4 5,000 100 5 ~40 Example 5 20,000 100 5 ~35
As shown in Table 1, a cell may be isolated in the pore as single
cell units by adjusting various conditions such as the amount of
number of second cells input, stirring speed, stirring time.
The present invention is not limited to the embodiments, and may be
prepared in different forms. Those skilled in the art of the
present invention can understand that it can be embodied in other
specific forms without departing from its spirit or essential
characteristics. Therefore, the described embodiments are to be
considered just as illustrative and not restrictive in all
respects.
DESCRIPTION OF REFERENCE NUMERALS
TABLE-US-00002 20: culture medium 30: porous membrane 31: pore 100:
first cell 200: second cell
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