JPH02276566A - Apparatus for culturing plant cell - Google Patents

Abstract

PURPOSE:To enable efficient practice of proliferation and classification with the size of cell conglomerates in a culture vessel by providing a construction for feeding a culture solution into the culture vessel, culturing plant cells, classifying cultured cell conglomerates through sieves provided in a culture solution passage in the vessel and taking out the cultured conglomerates. CONSTITUTION:A feed pipe 39 for a culture solution is provided on a culture vessel 20 for plant cells and the first sieve 21 and second sieve 23 are successively provided at an interval in a passage of the culture solution in the culture vessel 20. The culture solution is then fed from the feed pipe 39 into the culture vessel 20 and culturing is subsequently carried out while blowing a gas (e.g. air) from a gas blowing pipe 51 and stirring the culture solution. After completing the culturing, the feeding of the gas is stopped to open a valve 38 and discharge large cell conglomerates 25 together with the culture solution, A valve 40 is subsequently opened to take out the cell conglomerates 27 of the objective size together with the culture solution. A valve 42 is further opened to take out small cell conglomerates 29.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a plant cell culture device, and more particularly to a plant cell culture device capable of sorting into cell aggregates of different sizes.

[Conventional technology]

When producing useful substances using cultured plant cells, the cell growth conditions and the useful substance production conditions are often different, and two-stage culture is used.

In two-stage culture, as shown in Figure 5, cells that have been grown in advance in the first-stage culture tank under conditions suitable for cell proliferation are then transferred to the second-stage culture tank 9, where they are grown under conditions suitable for production. This is a method to produce the target useful substance by cultivating the target substance. In addition,
In the figure, 3 indicates the growth medium, 5 indicates the stirring blade, and 7 indicates the production medium.
The cells grown in the first stage of culture generally form clusters of several to several hundred cells. It is known that the ability of some plant cells to produce useful substances differs depending on the size of this cell aggregate. For example, AoM
, KinnersI! , ey et al.
In culturing andocyanin production from cultured cells of P. carota L+), it has been reported that agglomerates with a diameter of 63 μm or less have higher productivity than agglomerates with a diameter of 170 μm or more (A).

M,Kinnersfey et al,,F”1
anta, 149.200-204 (1980)).

In such a case, as shown in FIG. 6, in the first stage of culture, all the grown cells are removed from the culture tank 1, passed through a nylon or stainless steel sieve 15, and only the aggregates 11 of the desired size are removed. A possible method is to transfer it to the second stage culture tank 9.

13 is a cell aggregate smaller than the target size.

However, with this method, the cells must be taken out of the culture tank, and there are concerns about the effects of bacterial contamination.

[Problems to be Solved by the Invention] An object of the present invention is to solve the above-mentioned problems of the prior art and to provide a plant cell culture device that can efficiently perform jibut culture with a single device. be.

[Means to solve problems]

The present invention provides a culture tank, a means for circulating a culture solution in the culture tank, one or more sieves provided so as to block the flow path of the culture solution in the culture tank, and a cell classified by the sieve. It is characterized by having means for taking out each cell aggregate out of the tank.

[Example] An example of the present invention will be described with reference to the drawings. 1 to 3 are explanatory diagrams showing the apparatus of the present invention and its operating procedure.

The apparatus shown in FIG. 1 includes a culture tank 20, a culture solution supply pipe 39 provided at the top thereof, and a culture solution supply pipe 39 provided at intervals in sequence in the culture solution flow path (in the vertical direction) in the culture tank 20. The first
Removal of cell aggregates provided at cell aggregate storage locations on the sieve 21 and the second sieve 23, the gas blowing pipe 31 inserted into the bottom of the culture tank, and the first sieve 21 and the second sieve 23. It consists of tubes 35, 37 and a similar extraction tube 33 provided at the bottom of the culture tank. In FIG. 1, for example, when the purpose is to obtain cell aggregates with a size of 1 to 2 mm in the first stage of culture, the first sieve 21 is a mesh of 2 filters, and the second sieve 23 is a mesh of 1 mo+. It is attached to the culture tank 20, and gas (air,
Supply oxygen, nitrogen, etc.) and stir. As the culture continues, most of the cell aggregates 25 that have grown to 2 mm or more are on the first sieve 21 in the culture tank, and the cell aggregates 25 that are 1 mm or less are
Most of 9 collects at the bottom of the culture tank, and the target 1
A ~2 m agglomerate 27 collects on the second sieve 23 in the middle of the culture tank. The material of the sieve (hereinafter also referred to as mesh) may be any durable material such as nylon or stainless steel, but it is preferably one that is resistant to heat in order to sterilize the culture tank. Furthermore, there are no particular restrictions on the size of the mesh or the location of its attachment; however, the smaller the volume of the target size portion, the better the efficiency of agglomerate classification. It is more convenient to attach the mesh at an angle as shown in the figure when removing cell clusters. Although the figure shows one tilted to one side, it may also be shaped like a funnel to take out the cell aggregate from the center. When the culture is completed and the gas supply is stopped, the floating agglomerates settle and are separated into different sizes by meshes of different sizes as shown in FIG. Here, the valve 38 is opened (and the culture solution flows out, and along with the flow, 2 or more aggregates 25 are also discharged (Fig. 3)).Next, when the valve 40 is opened, 1 to 2 aggregates 25 are also discharged along with the culture solution. Cell clusters 27 of size 11 and below are discharged.Since the clusters of this size are the target size, they are transferred as they are to the second stage culture tank.Similarly, open the valve 42 and collect the clusters 29 of 11 and below. In addition, if the agglomerate 29 with 1III!1 or less is further cultured, the size of the agglomerate may become 1 to 2 mm, so it may be used for the next culture without being discharged.In this case, the conditioner For example, in the first stage of culture, agglomerates of 2ffiI11 or more and 2mm
When the purpose is to divide into the following agglomerates, as shown in FIG. May be stirred. The operations after completion of the culture are carried out in the same manner as in Example 1. In the figure, 40 is a culture tank, 47, 49 is a take-out tube, and 51 is a liquid streamline.

[Effects of the Invention] According to the present invention, it is possible to perform cell aggregation size classification as well as proliferation in a culture tank without using a classifier for cell agglomerate size.

[Brief explanation of drawings]

Figures 1, 2, 3 and 4 are explanatory diagrams showing the plant cell culture apparatus of the present invention, Figures 5 and 6, respectively.
The figure is an explanatory diagram showing a conventional plant cell culture device. 20... Culture tank, 21... First mesh, 23.・
・Second mesh, 25...first cell cluster (large), 2
7... Second cell aggregate (medium), 29... Third cell aggregate (small), 31... Gas blowing tube, 33.35.3
7...Cell cluster removal tube, L...Liquid level.

Claims (1)

[Claims] (1) A culture tank, a means for circulating a culture solution in the culture tank, one or more sieves provided to block the flow path of the culture solution in the culture tank, and cells classified by the sieve. 1. A plant cell culturing device characterized by having means for taking out each aggregate out of the tank.