JPH02219565A - Cell culture device - Google Patents

Abstract

PURPOSE:To remarkably improve the cell-culture efficiency of a cellculture device containing a culture medium having openable top by forming a gap between a lid and a main body to enable the flow of outer atmosphere and specifying the ratio of the culture area of the culture device to the cross-sectional area of the above gap. CONSTITUTION:The objective cell culture device contains a culture medium having openable top face covered with a lid and satisfies the formula A/G is 150-1000 wherein A is culture area of the culture device and G is the cross sectional area of a gap between the main body and the lid to allow the ventilation of outer atmosphere through the gap. The effect of the culture device is especially remarkable in a multi-well plate having a structure consisting of a number of assembled wells.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a cell culture device with improved cell culturability.

[Conventional technology]

Cell culture technology is currently frequently used and utilized in industry, and physiologically active substances such as drugs, hormones, and antibodies are produced. The cultivability of the cell culture vessel during cell culture, which is a preliminary stage of mass culture, is also very important for efficiency.

Conventionally, a Shear cloud multiwell plate or the like has been used as a small-scale culture vessel. In order to improve the culturability of cell culture vessels, methods such as covering the culture surface with physiologically active substances such as collagen and fibronectin, and performing special surface treatments have been proposed. However, these methods require special methods and techniques and are therefore difficult to apply to in-shell culture vessels.

Furthermore, these culture vessels have a structure in which a container with an open top is covered with a lid to prevent invasion of germs. Culture is 37
The culture is carried out in a culture tank at 100°C, and the humidity is adjusted to 100% in the presence of carbon dioxide gas to prevent changes in the pH in the culture medium. Therefore, in order to maintain ventilation with the outside of the incubator, a gap is generally provided between the lid and the container body.

[Problem to be solved by the invention]

The present invention aims to improve the culturability of cell culture vessels. As a result of studying the structure of the vessel, we found that the relationship between the culture area and the gap between the culture vessel body and the lid that allows for passage of outside air has a significant impact on cultivability. The present invention has been completed by discovering the The aim is to promote its application and utilization by increasing its effectiveness.

[Means to solve the problem]

The present invention is a cell culture device whose top surface can be opened, and the cross-sectional area (G) of the gap between the lid covering the top surface and the main body through which outside air can flow, and the culture area (A ) ratio A/
This cell culture vessel is characterized in that G is in the range of 150 to 1000.

In the present invention, the cell culture vessel whose top surface can be opened is a culture vessel constructed by covering a container with a lid, and is typically a petri dish, a multiwell plate, or the like. These have gaps that maintain ventilation with the outside during culture.

This aeration with the outside during culturing not only supplies oxygen to the inside, but also serves to keep the carbon dioxide concentration constant and the pH of the medium constant by aeration of carbon dioxide gas. However, on the other hand, it also has the opposite effect, such as causing a decrease due to evaporation of the medium.
Although this effect has not been given much attention, detailed studies have shown that it has a large effect on culturability, and it is possible to improve culturability by controlling this aeration.

The state of this ventilation is determined by the size of the gap created by the fit between the container body and the lid of the incubator, and the effect on culture performance can be expressed by the ratio to the culture area of the incubator. This ratio is expressed as the ratio A/G of the cross-sectional area of the gap (G) and the culture area (A). The larger this value is, the less aeration is, and the effect on cultivability is also high; however, if the value is very large, it will be the same as sealed culture, and during long-term culture, oxygen supply will be insufficient. This is not preferred, and a range of 150 to 1000 is appropriate for a culture period of several days to several weeks.

Additionally, this effect is a characteristic seen in general culture vessels, but 96.4
B. In multi-well plates such as 24 wells, the effect is even more remarkable.

The present invention will be explained below with reference to Examples.

〔Example〕

Multiwell plates and petri dishes having various culture areas (A) and gaps for ventilation (G) were prepared. After preparing each for cell culture and performing T-ray sterilization,
The medium was tested for evaporability and culturability. The incubator used in the test was as shown in Table 1, and the test methods for evaporability and culturability were as follows.

Method for measuring evaporation: Dispense a specified amount of pure water into each container, leave them in an incubator without humidity adjustment, and in an incubator adjusted to 100% humidity, then measure the amount of pure water remaining. , the residual rate (%) was determined.

For plates with a large number of wells, measurements were performed separately for the outer periphery and the inner part because the evaporation properties differed. The measurement results are shown in Tables 2 and 3.

Method for measuring culturability To evaluate culturability, the plate efficiency method (
PE method) Torimiting dileucidin method (LD method)
I did it. The measurement results are shown in Table 4.

・Plate E Proverb Shinshii method (PE method) Cells are HeLa
The medium used was MEM + 10% CS. A specified amount of cells were seeded in each container, and after culturing for about 10 days, the number of colonies formed was counted on the culture surface, and the ratio to the number of seeded cells was determined as plate efficiency (PE).

・Limity→Ningalyzing method (LD method) N5-1 cells were used, and RPMI-1640+10% FC3 was used as the medium. Cells were seeded at a specified concentration in each container (48 wells or 96 wells), the number of wells in which colonies were formed was counted, and the ratio of the number of seeded cells to all wells was determined as the colony production rate.

In addition, PE and colony production rate are numerical values that indicate what percentage of the seeded cells actually proliferated, and although they vary depending on the cell type and culture conditions, under these measurement conditions, the maximum is about 60% to 70%. be.

Regarding the relationship between the ratio of the culture area (A) to the gap CG (A/G ratio) and the evaporability of the medium, there is a clear correlation with the residual rate measured in an incubator without humidity adjustment, and the A/G ratio Containers with high levels of residual water have a high residual rate.

In an incubator with 100% humidity control in which culture is actually performed, differences appear in the outer wells of long-term incubators.

These characteristics affect the culturability of cells, and in containers with a low Al6 ratio (N114.7.8.9.10.11), PlateF? The colony formation rate becomes smaller. Samples with an A/G ratio of 150 or more (Nllll, 2.
3.5.6), the culturability is improved and the plate efficiency or colony production rate shows a high value of about 60% or more.

Although these effects can be seen in petri dishes, the effects are particularly remarkable in multiwell plates.

In addition, an incubator with an A/G ratio of 1000 or more (13) is close to a sealed culture and has very little ventilation, so it has high cultivability (also, long-term [effects of the invention]) according to the present invention. It is possible to easily create a cell culture vessel with excellent culturability, and the culturability and viability of cells can be reproduced (obtained, providing a cell culture vessel with optimal performance as a cell culture vessel. can do.

Claims (1)

[Claims] (1) A cell culture device with an open top culture surface,
It is characterized in that the ratio A/G of the cross-sectional area (G) of the gap between the lid covering the top surface and the main body through which outside air can circulate and the culture area (A) of the incubator is in the range of 150 to 1000. Cell culture vessel.