JP2005137319A - Cell culture vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To disclose a cell culture vessel capable of fixing inoculated cells completely to the surface or the inner part of a cell culture carrier, and provide a cell culture vessel to enable easy quantitative evaluation of a plane or three-dimensional culture carrier as an anchorage material and easy observation of the process of growth of the cell from outside. <P>SOLUTION: The culture vessel is composed of a cylindrical outer cylinder 1 and a cylindrical inner cylinder 2 having an inner space and fittable to the outer cylinder. The vessel is further provided with a bottom plate 4 insertable into the inner space of the outer cylinder and a packing 3 to prevent the leakage of liquid through a gap between the bottom plate and the outer cylinder. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an apparatus for culturing cells using a planar or sheet-like cell culture carrier (Scaffold). More specifically, the outer edge of the cell culture carrier is fixed, and the number of cells used for the evaluation. The present invention relates to an incubator that can be fixed to a cell culture carrier without damaging.

  As technological innovation related to regenerative medicine technology progresses in recent years, culture using a cell culture carrier that becomes a scaffold for the growth, differentiation, and organization of cells has become important. As a technique for immobilizing or encapsulating cells in a cell culture carrier, a method of encapsulating cells in a cell culture carrier using a gel such as collagen or alginic acid, or absorbing a medium containing cells by capillary action and immobilizing the cells in the process There are known a method of making it, a method of simply placing it on the bottom of a cell culture vessel, and a method of immobilizing with an appropriate weight.

  When the cells grow and differentiate / organize, in the above culture method, the seeded or proliferated cells leak out of the culture carrier or enter the back of the culture carrier. It was difficult to take out well.

  In addition, regarding each process in which cells grow and differentiate on or in a culture carrier, the growth state of the cells, the type and amount of extracellular matrix produced by the cells, the state of organization, etc. are observed in detail or There is a growing need for evaluation.

Regarding a container for culturing cells on a film-like material, for example, JP-A-52-38082 (Patent Document 1) describes a culture container in which a gas-permeable film is tightly attached to the bottom. Yes. As this film, only a very thin film that allows gas permeation but not liquid permeation can be used. For example, a cell culture that allows liquid to permeate through a concavo-convex molded body such as a freeze-dried sponge or a non-woven cell culture carrier. It cannot be used for culture using a carrier.
JP 52-38082 A

  The problem to be solved by the present invention is to disclose a culture vessel that can be fixed to the surface or inside of a cell culture carrier without leaving the seeded cells, and further to a planar or three-dimensional culture carrier. It is to disclose a cell culture device that makes it easy to observe the process of cell growth from the outside so that quantitative evaluation of how excellent the cell scaffold is can be easily performed. .

  The culture container of the present invention comprises a cylindrical outer cylinder (1) and a cylindrical inner cylinder (2) that has a lumen and can be fitted into the outer cylinder, and can be inserted into the lumen of the outer cylinder. It is an incubator having a bottom plate (4) and a packing (3) for preventing liquid leakage between the bottom plate and the outer cylinder.

  Moreover, it is a cell culture device which has a cell culture support | carrier (5) between the bottom part of this inner cylinder (2), and a baseplate (4).

  By using the cell culture device disclosed in the present invention, the seeded cells can be encapsulated or fixed on the surface of the cell culture carrier without any damage, so that the cell characteristic evaluation or cell culture carrier characteristic excellent in quantitative properties can be obtained. Can be evaluated. It is also possible to directly observe the growth state of the cells at that time.

  Hereinafter, the present invention will be described in detail. In addition, these Examples etc. and description illustrate this invention, and it cannot be overemphasized that it may belong to the category of this invention.

The configuration of the present invention will be described with reference to the drawings.
In FIG. 1, a cylindrical outer cylinder (1) and an inner cylinder (2) may be made of resin, metal or glass, and an observation lumen (6 ) And the lumen (7) for holding the liquid medium is open. The outer cylinder has a step for placing a packing (3) for preventing the liquid medium from leaking, and the outer cylinder (1) and the inner cylinder (2) have a thread groove so that the inner cylinder becomes an outer cylinder. The end of the inner cylinder can be screwed, and the cell culture carrier (5) and the bottom plate (4) can be crimped and fixed.

  A packing (3) is inserted between the outer cylinder (1) and the bottom plate (4) to prevent leakage of the liquid medium. As the packing (3) for preventing liquid leakage, any rubber-made packing may be used, but a silicone or fluororubber packing having low cytotoxicity is preferably used.

  The flat plate serving as the bottom of the liquid medium, that is, the bottom plate (4) is preferably transparent, and a flat plate made of glass or transparent resin is preferably used. The cell culture carrier (5) is placed on the bottom plate (4), and is crimped from above using the inner cylinder (2). The inner cylinder (2) has a lumen for holding a liquid culture medium, and cells can be observed from this lumen.

  The cell culture carrier (5) can be prevented from leaking from the side of the carrier when the cells are seeded from above by crimping the circumferential edge of the cell culture carrier (5) from above, and even more uniform from the top. Since the end of the cell culture carrier is pressed, the surface of the cell culture carrier facing the lumen (7) can be approximated to a horizontal shape, and in particular, the surface of a molded body with many irregularities such as a freeze-dried sponge can be kept horizontal. Is possible.

  The material of the outer cylinder (1) and the outer cylinder (2) may be made of resin, metal or glass, but it is made of stainless steel because it is easy to obtain, process and sterilize. And Teflon (registered trademark) resin is preferred. Among them, austenitic stainless steel is preferable because it is suitable for autoclave (steam heating) sterilization frequently used in research and development work. As a specific example of stainless steel, stainless steel made of SUS304 or SUS316 is preferably used.

  When culturing cells using a molded stainless steel body, it is preferable that the surface of the stainless steel be subjected to an immobilization treatment. Any known method may be used for the immobilization treatment, but it is preferable to wash with an acid aqueous solution of about 0.01 to 1N, more preferably an acidic aqueous solution containing phosphoric acid or phosphorous acid. It is good to use as a cleaning agent.

  The cell culture carrier used in the present invention is preferably in the form of a plane, a sheet, or a sponge. More specifically, the carrier having a thickness of 10 mm or less is preferred, and a carrier having a thickness of 3 mm or less is more preferred. If the cell culture carrier is thicker than this, it is not preferable because it is difficult to maintain airtightness by pressure bonding. Moreover, in order to make the crimping more reliable, a sealing material such as silicon rubber or packing is appropriately used.

  A film-shaped culture carrier can also be preferably used. The film surface may be uneven or smooth. A foam or a sheet-like molded body is also preferably used. Moreover, even if it is a porous body having continuous pores, a sheet made of a fiber such as a nonwoven fabric, or a mesh made of monofilament, it can be evaluated using the incubator of the present invention.

  The lumen of the outer cylinder serves as a window hole for observing the growth state of the cell, and the growth of the cell can be observed using an inverted microscope.

  FIG. 2 illustrates another embodiment of the culture vessel of the present invention. The cylindrical outer cylinder (1 ′) and inner cylinder (2 ′) may be made of resin, metal, or glass, and are used for observation and holding a liquid medium in the center. Is open (7 '). The outer cylinder (1 ′) has a bottom portion on which a packing (3 ′) for preventing the proliferated cells from wrapping around the back surface of the bottom plate is placed, and the outer cylinder (1 ′) and the inner cylinder (2 ′) are threaded grooves. The inner cylinder can be screwed into the outer cylinder, and the end of the inner cylinder can be crimped and fixed to the cell culture carrier (5 ′) and the bottom plate (4 ′).

  A packing (3 ') is inserted between the outer cylinder and the bottom plate to prevent leakage of the liquid medium. As the packing (3 ') for preventing liquid leakage, any rubber-made packing may be used. However, a silicone or fluororubber packing having low cytotoxicity is preferably used.

  The flat plate that is the bottom of the liquid medium, that is, the bottom plate (4 ') is preferably transparent, and a flat plate made of glass or transparent resin is preferably used. A cell culture carrier is placed on the bottom plate (4 '), and crimped from above using the inner cylinder (2'). The inner cylinder (2 ') has a lumen for holding a liquid medium, and cells can be observed from this lumen.

  The cell culture carrier (5 ′) can be prevented from leaking from the side of the carrier, which occurs when cells are seeded from above, by crimping the end of its circumference from the top. Since the end is pressed evenly, the surface of the cell culture carrier facing the lumen (7 ') can be approximated to a horizontal shape, and in particular, the surface of a molded body with many irregularities such as a freeze-dried sponge can be kept horizontal. It is possible.

  The material of the outer cylinder (1 ′) and the outer cylinder (2 ′) may be made of resin, metal or glass, but it is observed from the outside, particularly from the bottom of the outer cylinder. To make this possible, a transparent resin or glass is desirable. Stainless steel or Teflon (registered trademark) resin is preferred from the viewpoint of easy availability and processing and sterilization. Among them, austenitic stainless steel is preferable because it is suitable for autoclave (steam heating) sterilization frequently used in research and development work. As a specific example of stainless steel, stainless steel made of SUS304 or SUS316 is preferably used.

  When culturing cells using a stainless steel culture vessel, it is preferable to immobilize the stainless steel surface. Any known method may be used for the immobilization treatment, but it is preferable to wash with an acid aqueous solution of about 0.01 to 1N, more preferably an acidic aqueous solution containing phosphoric acid or phosphorous acid. It is good to use as a cleaning agent.

  If the culture vessel is made of transparent resin or glass, cell growth can be observed using an inverted microscope, and if it is a stainless steel culture vessel, it can be observed from the lumen of the inner cylinder. it can.

  In either case of FIG. 1 or FIG. 2, the upper part of the inner cylinder may be closed or may be closed by a lid.

  The details of the present invention will be described more specifically by the following examples. However, the present invention is not limited to these examples.

[Example 1]
As shown in FIG. 1, a fluororubber packing (3) and a glass plate bottom plate (4) are formed on a step portion of a cylindrical outer tube (1) having a lumen (6) as a window hole for observation. And a cast film of polylactic acid, ie, a cell culture carrier (5). A cylindrical inner cylinder (2) having an observation window hole, that is, a lumen (7) and having a thread groove on the outer peripheral portion was inserted, and a cast film as a cell culture carrier was pressure-bonded from above. The inner diameter of the cylindrical inner cylinder (2) was 24 mm.

Chondrocytes were cultured by placing primary chondrocytes collected from rabbit cartilage and liquid medium into the lumen (7) for 5 minutes. The number of cells used for the culture was about 2 × 10 ⁵ . The culture was carried out under conditions of 5% carbon dioxide concentration and 37 ° C., and the liquid medium was changed as appropriate. Two weeks later, cell number proliferation and extracellular matrix production could be observed. No cells were observed on the lower part of the cast film as the cell culture carrier, that is, the side facing the bottom plate (4), and it was confirmed that the cells did not leak and were immobilized on the surface of the cast film as the cell culture carrier.

  The incubator disclosed in the present invention is an incubator useful for evaluating planar or sheet-like cell culture carriers and confirming the growth status of cells in cell culture carriers, and quantitative evaluation of cells. It is possible to quantitatively evaluate the extracellular matrix calculated by the cells. In the field of regenerative medicine, this incubator can be an important tool for evaluating a culture carrier (Scaffold) for cell culture, and evaluating the growth state and growth process of cells in Scaffold.

2 is a cross-sectional view of the culture container of Example 1. FIG. Sectional drawing of the culture container of another aspect from the culture container of Example 1. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Outer cylinder 2 Inner cylinder 3 Packing 4 Bottom plate 5 Cell culture carrier 6 Inner cylinder lumen 7 Inner cylinder lumen 1 'Outer cylinder 2' Inner cylinder 3 'Packing 4' Bottom plate 5 'Cell culture carrier 7' lumen

Claims (4)

  A bottom plate (1) comprising a cylindrical outer cylinder (1) and a cylindrical inner cylinder (2) which has a lumen and can be fitted or screwed into the outer cylinder, and is inserted into the lumen of the outer cylinder ( 4) and an incubator having a packing (3) for preventing liquid leakage between the bottom plate and the outer cylinder.   The cell culture vessel according to claim 1, further comprising a cell culture carrier (5) between the bottom of the inner cylinder (2) and the bottom plate (4).   The incubator according to claim 1 or 2, wherein the inner cylinder (1) and the outer cylinder (2) are made of austenitic stainless steel subjected to non-moving object processing.   The evaluation method which implements evaluation of this cell culture support | carrier using the incubator of Claims 1-3.

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