JP5268383B2 - Molded body for forming a plurality of microdroplets and molding method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a formed article capable of producing a cell array composed of different kinds of cells on the same plane without using a complicated apparatus constitution while keeping the surviving condition of the cells. <P>SOLUTION: The formed article has the first, the second and the third planes parallel to each other, a plurality of independent capillaries passing through the first and the third planes and a region between the first and the second planes and perpendicular to the first and the second planes, wherein all capillaries contained therein are parallel to each other and the distance between an arbitrarily selected capillary and all remaining capillaries in the parallel planes between the second plane and the third plane increases toward the third plane. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a resin molded body for arranging micro droplets in a lattice pattern.

  In recent years, interest has been focused on the analysis of the mechanism of cell-cell interaction such as what kind of interaction works between different cells. In order to examine such interaction between cells, it is first necessary to arrange different kinds of cells in the same container at high density.

  However, as disclosed in Patent Document 1, a conventional method of arranging a large number of micro containers is used to arrange cells, but this method is used when different types of cells are arranged in a high density in the same container. I can't.

  Moreover, as seen in Patent Document 2, a technology for arranging a large number of oligonucleotides in a single container at a high density by applying a semiconductor process has already been established. There is no known method for arranging them in the same container at a high density.

Further, as seen in Patent Document 3, a technique for detecting an electrophoresis band using a capillary array that can inject and move a plurality of samples containing nucleic acids or proteins is known. A method for producing an array of heterogeneous cells using such a plurality of capillaries is not known.
JP 2005-059524 A Japanese Patent No. 3495702 Japanese Patent Laid-Open No. 2002-131282

  In order to form a high-density heterogeneous cell array on a flat surface using capillaries, it is necessary to introduce a different liquid for each capillary, so in order to handle various liquid samples to form a heterogeneous cell array It is desirable that the configuration of the apparatus is simple. It is also necessary to maintain environmental conditions such as physiological conditions and temperature of the cells.

  Accordingly, an object of the present invention is to provide a molded body that can produce a cell array composed of heterogeneous cells on the same plane while maintaining the cell survival conditions without taking a complicated apparatus configuration.

  Still another object of the present invention is to provide a method for producing a molded body for producing an array of heterogeneous cells.

The method for manufacturing a capillary array according to the present invention includes the steps of forming an aggregate formed by bundling a linear material multiple, pouring a resin material uncured wherein the collection, and curing the resin material, the Removing a plurality of linear substances and obtaining a resin molded body having a plurality of penetrated capillaries, and a method for molding a capillary array having a plurality of capillaries,
The capillary array has a first portion in which the distance between the capillaries becomes closer toward the other end from the ends of the plurality of capillaries;
A second portion connected to the first portion, the capillaries being arranged in parallel, and two different focusing structures;
A first resin molding step of pouring an uncured resin material into a portion of the assembly corresponding to the first portion of the capillary array, and curing the resin material;
A second resin molding step in which an uncured resin material is poured into a portion of the assembly corresponding to the second portion of the capillary array, and the resin material is cured;
A method for forming a capillary array, comprising:

  In the molded article of the present invention, a fine heterogeneous cell array can be easily arranged on a plane. In addition, since it has a structure that does not have a complicated mechanism and can introduce different liquids for each capillary, it is possible to produce a cell array composed of various cells without requiring complicated operations and maintenance. it can. Furthermore, since the microdroplet array can be formed without applying energy such as heat, the cell array can be formed in a state where the cells are alive.

  The present invention relates to a molded body capable of arranging a plurality of minute droplets at high density on the same plane without taking a complicated apparatus configuration by a simple resin molded body. Arranging on the same plane means that a plurality of micro droplets are arranged on the same plane without being separated by partitions such as containers and walls. Since the molded body has a structure capable of forming microdroplets containing cells, it can be suitably used for preparing an array of heterogeneous cells.

  FIG. 1 shows an example of a molded body 1 according to the present invention. A plurality of capillaries 3 have a capillary array structure penetrating from the upper part to the lower part, and a plurality of micro droplets 2 can be imprinted and formed at a high density.

  Based on FIG. 2, the molded body will be described in detail. The molded body has a first surface 4, a second surface 5, and a third surface 6 that are parallel to each other. The first surface is a surface that discharges micro droplets and imprints on a flat surface (micro droplet imprinting surface), and the third surface is a surface that introduces liquid that forms micro droplets into the molded body ( The second surface is a cross section of the molded body existing in parallel with the first surface and the third surface. A plurality of independent capillaries 3 penetrating the first and third surfaces 6 are formed in the first and second surfaces 5 in a region between the first and second surfaces 5 (hereinafter also referred to as a first region). And all the capillaries 3 are arranged in parallel. Further, in the region between the second surface 5 and the third surface 6 (hereinafter also referred to as the second region), the capillary 3 has a distance in the parallel plane between the capillary 3 selected arbitrarily and all the remaining capillaries. It has a structure that moves away as it approaches the third surface 6.

  The space between the capillaries in the first region corresponds to the distance between the microdroplets formed on the bottom surface of the microplate. Therefore, the interval needs to be narrow enough to form a high-density microdroplet array. Therefore, a structure in which a plurality of capillaries are converged (capillary array) is formed in the first region. The dimension in the longitudinal direction of the first region is preferably designed so as to correspond to the depth of the microplate to be formed with the microdroplet array.

  The capillaries in the second region are not particularly limited as long as the capillaries have structures that move away from each other as they approach the third surface, but the gap between the capillaries exposed on the third surface is manually controlled. It is preferable that it is wide enough to supply liquids individually by dispensing. As a result of being able to introduce different liquids into the respective holes by manual operation, different liquids can be included in the microdroplet pattern imprinted from the imprinting surface. Therefore, it can be suitably used for forming a micro liquid pattern composed of micro liquid droplets each containing different cells.

  The capillary array structure of the molded body can be formed by molding a linear substance as a mold with a resin or the like. By using a thin and non-rigid material as a linear material for a capillary mold, the capillary array structure composed of the first region and the second region can be easily molded using a two-step molding step. Can do. Specifically, an example of a method for producing a molded body will be described with reference to FIGS. FIG. 3A shows a process of forming a linear assembly having the converging portion 8. A member of the converging part 8 is a single-wafer member 7b having a plurality of through holes at high density, and the linear substances 9 are bundled. Further, one end of the linear substance forming the linear assembly is passed through a single-wafer member 7a having through holes that are low in density to allow manual dispensing operation. As shown in the figure, a linear assembly can be formed between the sheet member 7a and the sheet member 7b. This linear assembly is a region having no focusing structure. Next, FIG. 3B shows a process of forming a linear assembly in a region having no focusing structure. The resin is poured into the molds 13a and 13b that can be halved in FIG. 4 to form a linear assembly. In this step, the molded body 10 is formed. 3C, after the molded body 10 is molded, the converging portion 8 is moved to the other end side of the linear assembly (opposite side through the sheet member 7a) to focus. The process of forming the linear aggregate | assembly which has a structure is represented. The focusing structure is formed between the position of the focusing unit 8 before movement and the focusing unit 8 after movement. Subsequently, FIG. 3D shows a process of pouring the resin to the converging part after movement and molding a region having the converging structure. A molded body 11 is formed. After all the resin is cured, the member (including the linear substance 9) that becomes the mold is removed. As the resin, it is preferable to use a material having moderate elasticity after curing, such as PDMS shown in the examples, because liquid leakage at the time of stamping can be prevented. In addition, it is preferable to use a gas-permeable material such as PDMS because it is suitable for the culture environment. Further, in the process of molding either the molded body 10 or the molded body 11, a Si wafer that has been finely processed can be used as a part of the mold.

  As shown in FIG. 4, the half molds 13 a and 13 b are combined with screws 12. The converging part 8 may be provided with holding parts 14a and 14b on the mold side. The holding portions 14a and 14b can be shaped to hold the sheet member 7a and the sheet member 7b. The linear substance 9 can be a thread or a microtubule. For example, nylon thread, glass fiber thread, extra fine vinyl tube, extra fine glass tube, etc. can be mentioned. As the sheet member forming the converging portion 8, for example, a stainless steel plate subjected to through etching, a Si wafer, a glass plate, a cloth such as nylon or Teflon woven in a mesh shape with a certain pore size can be used.

However, in order to prevent liquid mixing, when providing a convex portion in the capillary hole of the first surface 4,
It is desirable that the Si wafer is used as a single-wafer member for forming the converging portion and processed by through etching by a fine processing method such as DEEP-RIE. An example of the convex shape is shown in FIGS. Further, (A) shows a cross section 15 of the Si wafer. Resin is poured and a convex part is formed.

  The molded body that can be conveniently produced in the present invention is preferably a capillary having a diameter of several tens to several hundreds of microns. Further, the inside of the capillary tube may be treated and coated with hydrophilicity or hydrophobicity according to circumstances.

  From the aspect of mastering (when pipetting the liquid into the tube), it is possible to easily put the liquid by coating the inside of the tube with a hydrophilic property. In general, considering a capillary tube with a radius r, in order to move the fluid (density ρ, liquid column h) in the tube by applying pressure P from one end of the tube, P> 2T cos θ / r − It should be ρgh. Where T is the surface tension, θ is the contact angle between the liquid and the tube, and g is the gravitational acceleration. Therefore, if θ is smaller than 90 °, the pressure required to move the liquid in the tube requires a larger value as r 1 becomes smaller.

  Moreover, the molded body of the present invention is used for the purpose of stamping and forming on the bottom surface of a microwell plate or the like. For example, when imprinting on a 96-well plate, the size of the first surface 4 must be less than 9.3 mm. For a 48-well plate, it must be less than 11.0 mm. Moreover, it is desirable that the molded body of the present invention is easy to dispense. Accordingly, in addition to the size of the first surface 4, the pitch of the capillaries penetrating the third surface 6 can be a distance that facilitates the dispensing operation. This distance can be automated by using a 2.25 mm pitch, which is a standard for 1536 well plates, for example.

(Si wafer processing)
A Si wafer is used as the sheet member 7a that forms the first surface of the molded body and is processed by the DEEP-RIE method to form a concave portion on the Si wafer to form a convex shape. First, a mask pattern having a convex shape is drawn to produce a mask. Two types of masks are produced: a pattern etched from the front surface of the Si wafer and a pattern etched from the back surface.

  Thick film resist AZ4903 (AZ Electronic Materials Co., Ltd.) is spin coated, exposed and developed on the Si wafer surface. The wafer is put into an ICP etching apparatus (MUC-21: manufactured by STS) and etched approximately vertically by about 350 μm. The ratio of etching: passivation time is 7: 2. After the etching is completed, the resist is removed and washed with acetone. Next, the resist AZ4903 is spin-coated on the back surface of the wafer, and after alignment with the etching pattern on the surface with a double-sided aligner, exposure and development are performed. After performing a taping process for preventing leakage on the surface side, the wafer is put into the ICP etching apparatus, and etching is performed until it penetrates. The wafer is diced to a desired size.

  In the case of a thick film resist, it is used when the pattern is about 60 μm, and in the case of a finer mask pattern larger than that, it is replaced with a metal mask by a lift-off process. The metal is deposited about 2000mm of chrome.

A through-hole is also formed in the Si wafer used as the single-wafer member 7b forming the converging portion, and dicing to a desired size is performed.
The interval between the through holes is 2.25 mm, which is a standard for 1536 well plates, and the sheet member 7b is 250 μm.

(Formation of linear assembly with converging part)
FIG. 6 shows a photograph of a linear assembly having a converging portion and a mold for storing it. As the linear material, nylon thread of 100 μm is used. Nylon thread is passed through the upper and lower Si processed wafers after through etching and dicing using a magnifying glass (upper photo).

(Mold linear assembly)
PDMS (polydimethylsiloxane) resin (SYLGARD 184: Dow Corning Co., Ltd.) is mixed 10: 1 with the attached polymerization initiator and centrifugally degassed. The linear assembly formed above is placed in a mold, and PDMS resin is poured. Mold overnight in a cool dark place.

(Movement and molding of converging part)
The cured PDMS resin is removed from the mold, and the converging part is moved backward. The mold is put in the mold again, and the previous PDMS resin is poured into the space thus formed.

  FIG. 7A shows the molded body after molding. FIG. 7B shows a tip portion having a convex shape. FIG. 7C shows a photograph of the processed surface of the Si wafer that becomes the convex mold. A convex shape is formed by pouring resin into the concave portion of the photograph.

  The present invention is an important tool for analyzing the interaction between different types of cells. In the molded body of the present invention, normal culture other than hanging drop culture may be performed. It can also be used to simply create spot arrays of different liquids.

An example of the molded object of this invention is represented. The molded object explanatory drawing of this invention is represented. Explanatory drawing of the molded object production method of this invention is represented. An example of the molded object casting_mold | template of this invention is represented. An example of the convex shape is shown. A photograph of a linear assembly with a converging part and a mold to be stored. (A) Photograph of the molded body. (B) Photo of the tip of the convex shape. (C) Si wafer processing surface photograph used as a convex-shaped mold.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Molded body 2 Micro droplet 3 Capillary 4 First surface 5 Second surface 6 Third surface 7a, 7b Single-wafer member 8 Converging part 9 Linear substance 10 Molded body (process B)
11 Molded body (Process D)
12 Screws 13a, 13b Half molds 14a, 14b Holding part 15 Si wafer cross section

Claims (3)

Forming an aggregate formed by bundling a linear material multiple,
Pouring an uncured resin material into the assembly and curing the resin material;
Removing the plurality of linear substances and obtaining a resin molded body having a plurality of penetrated capillaries, and a method for molding a capillary array having a plurality of capillaries,
The capillary array has a first portion in which the distance between the capillaries becomes closer toward the other end from the ends of the plurality of capillaries;
A second portion connected to the first portion, the capillaries being arranged in parallel, and two different focusing structures;
A first resin molding step of pouring an uncured resin material into a portion of the assembly corresponding to the first portion of the capillary array, and curing the resin material;
A second resin molding step in which an uncured resin material is poured into a portion of the assembly corresponding to the second portion of the capillary array, and the resin material is cured;
A method for forming a capillary array, comprising: The molding method according to claim 1, wherein the plurality of capillaries are respectively connected to the through holes of the Si wafer having a plurality of through holes at the end of the first portion. The resin material, molding method according to claim 1 or 2 is a PDMS (polydimethylsiloxane).