KR20050020964A - Cell culture device - Google Patents

Abstract

The present invention relates to an apparatus for cell culture. The present invention includes a chamber (100) for containing a cell (10) comprising at least one liquid injection conduit (121, 122, 123, 124, 125, 126), the chamber 100 being A cell culture apparatus configured to freely contact a surface of a liquid with air. The invention comprises a collector member 40 which can form at least one liquid bridge with the free surface of the liquid and comprises a mouthpiece 43 positioned at the level of the free surface of the liquid contained in the chamber 100. Characterized in having a. The mouthpiece 43 described above is maintained in a vacuum to absorb the air flow that moves the surface film of the liquid via the liquid bridge.

Description

Cell culture device {CELL CULTURE DEVICE}

The present invention relates to a cell culture apparatus capable of treating various media while avoiding hand handling.

French Patent No. 2 659 347 (published Sep. 13, 1991) discloses a cell culture device comprising a chamber for containing cells. The chamber includes a horizontal support formed by two glass plates spaced apart side by side to form a slot between the slots having a width smaller than the diameter of the cell. The cells to be processed are placed on slots. This chamber is for receiving liquid culture or pulse media. Various liquid media are continuously injected into the chamber via a tube located above the wall supporting the cells. Some of the fluid injected into the chamber is evacuated by one or more tubes located below the wall. The other part of this fluid is discharged by overflow.

In this device, cells are retained on the slot by reduced pressure caused by suction of the fluid medium by suction through the discharge tube.

Such devices are particularly for the cultivation of oocytes or hatched eggs or embroiders and the like.

In particular, it can be used for the activity of experimental oocytes, which is necessary for the correct continuous development of embroiders. To induce this activation, the culture medium is injected into the chamber and the activated cells are placed in this culture medium. The iron culture medium is then discharged and replaced with a pulse medium containing calcium ions (Ca ²⁺ ) at the same time. Once the culture medium is completely drained and replaced with a pulsed medium, the cells are subjected to electric field pulses, which causes the instantaneous electron permeation of their plasma membranes and the penetration of calcium ions (Ca ²⁺ ) into the cells. The pulse medium is then discharged in turn and replaced with culture medium. This step is repeated several times so that the cells receive a controlled series of calcium pulses that promote their activation.

The device is characterized in that the cells can be treated with various media while avoiding the handling of the cells.

However, a problem with this type of device is that it is relatively long to change from one medium to another, which limits the frequent change of medium in the chamber.

For example, when the device is used for cell activation, a minimum time (40 seconds) for injection of the pulse medium is needed to replace the culture medium with the pulse medium. In fact, this minimum time allows the cells to be sufficiently washed with a pulse medium.

If this minimum wash time is not observed, the pulse medium may contain residual ions coming from the culture medium. When an electric field is applied, these ions can generate transmembrane ion currents that can destroy cells.

Another problem associated with washing is that long exposure of cells in pulsed media with low ionic strength breaks the cell's equilibrium and results in deleterious effects. Thus, to ensure cell survival, it is necessary to reduce the washing time of the cells.

One object of the present invention is to provide a cell culture apparatus capable of rapidly replacing a medium in which a cell is located.

It can be seen that the discharge of fluid due to overflow is a non uniform phenomenon. In fact, during the injection of the liquid into the chamber, the free side of the liquid rises above the top plane of the lid, which takes the form of a convex crescent. This phenomenon is related to the surface tension occurring at the liquid surface and the wettability of the vertical wall of the liquid cover. The difference in liquid level above the lid edge must first reach a threshold at which the equilibrium between the force associated with surface tension and the force associated with gravity is broken. This breakdown results in excess liquid flowing over the cover wall.

The discharge of the liquid by overflow occurs discontinuously and unpredictably. In particular, the medium to be replaced in the step of replacing one medium with another is not discharged. This discharge is not satisfactory because the washing of the cells is not satisfactorily guaranteed during this alternative phase.

Moreover, another problem is that this discontinuous flow creates a continuous shock wave in the liquid. These shock waves move the cells onto the support.

The cells tend to move along the slots of the support member towards the center of the cover in which they are present. They are compressed into each other. Reducing the spacing between cells alters the efficiency of washing at the periphery of each cell.

Finally another problem is that the interface between the culture medium and the surrounding gas is a surface development site. The atomic or molecular film forms the free surface of the liquid. These films do not change when one medium is replaced with another in the chamber.

When the incubator is used to apply electric field pulses to the cells, the films can form a conductive bridge between electrons. These conductive bridges can form short circuits between electrons. As a result, the efficiency of the applied electric field drops significantly.

In order to solve the above problems, the present invention provides a cell culture apparatus including a chamber for containing a cell, the cell culture apparatus including at least one liquid injection conduit, wherein the surface of the liquid contained in the chamber is in free contact with air. An air flow that can form at least one liquid bridge with the free surface of the liquid and is located at the level of the free surface of the liquid contained in the chamber and maintained at a low pressure to move the surface film of the liquid via the liquid bridge. It provides a cell culture apparatus comprising a collector member comprising a mouthpiece to absorb the.

The device of the present invention makes it possible to form a crescent shaped liquid bridge between the free surface of the liquid and the collector member. This crescent shape controls the surface tension at the surface of the liquid contained in the chamber by the equilibrium effect.

Such a device allows for the continuous removal of excess fluid from the chamber by the flow of air and thus to maintain a constant level of liquid.

It is also possible to permanently renew the surface film of the liquid in the chamber.

In one embodiment of the invention, the collector member is positioned on the rim of the chamber wall with its mouthpiece stepped back from the wall.

Preferably, a portion of the rim located in front of the mouthpiece of the collector member is covered with a hydrophilic material.

Preferably, the mouthpiece is positioned stepped back from the wall at a distance d of 10-30 microns.

Preferably, the mouthpiece of the collector member is elongated and extends longitudinally along the edge of the wall.

In a preferred embodiment of the invention, the opening has a length of 2-4 mm. In a preferred embodiment of the invention the opening has a height of 0.15-0.30 mm.

In one embodiment of the present invention, the mouthpiece of the collector member has a rectangular shape.

The device of the invention comprises several members located along the edge of the chamber wall.

The invention also provides a method for regulating the level of liquid contained in a chamber for containing a cell comprising at least one liquid inlet conduit, wherein the surface of the liquid contained in the chamber is configured to make free contact with air. At least one liquid bridge can be formed with the free surface, and because the flow of air is by suction around the liquid bridge, this flow of air moves the surface film of the liquid via the liquid bridge. A liquid level control method.

Other features and characteristics will be described from the following description given as a non-limiting example with reference to the accompanying drawings.

1 is a diagram of a cell culture apparatus according to an embodiment of the present invention.

Figure 2 is a detailed view of the collector of the apparatus of Figure 1;

3 is a cross-sectional view of the collector of FIG.

In FIG. 1, the cell culture apparatus has a chamber 100 including wall portions 11, 12, 13 defining a lid 20, intended to include a liquid medium. The lid is filled with a liquid medium corresponding to the ongoing cell treatment step. In the lid 20, the horizontal support member is formed and positioned by two glass plates 101, 102 side by side. The glass plates 101 and 102 are wrapped and held by the side walls 11 and 12 of the chamber. These glass plates 101 and 102 are respectively spaced apart from each other to form rectangular slots 103 having a width smaller than the diameter of the oocytes 10 to be processed therebetween.

The chamber 100 also includes electrodes 111, 112 extending longitudinally to both sides of the slot 103.

The culture liquid is introduced into the upper portion of the lid 20 by a series of conduits, ie mouthpieces 121, 122, 123, 124, 125, 126, which are positioned at regular intervals along the walls 11, 12, 13. Moreover, some of the liquid contained in the chamber is discharged by the discharge conduit 104 located at a level lower than that of the support member. The liquid stream attaches oocytes to the slots by low pressure. The free surface of the liquid contained in the chamber 100 forms a convex molecular film 30. The film 30 consists of an oriented molecular arrangement in which the hydrophilic part is located towards the fluid and the hydrophobic part is located towards the outside air.

The incubator includes a collector 40 positioned on a horizontal rim 20 of the wall of the lid 20 to remove the molecular film 30 and absorb excess liquid contained in the lid.

Symmetrically, the device may comprise a collector located on the rim 22 or 23 of the wall portion 12 or 13 (indicated by the dashed lines).

These collectors 40 may be located at regular intervals around the cover 20.

FIG. 2 is a detailed view of the collector 40 of the cell culture apparatus of FIG. 1. This collector 40 enters the distance d from the wall and is located on the rim 21 of the chamber wall 11. The collector includes a hollow body portion 41 having a rectangular opening extending along the wall portion 11. This opening has a length L of 2 mm and a height l of 0.2 mm.

Two fluid bridges 51 and 52 are formed between the liquid surface 30 included in the lid 20 and the vertical edge of the opening 42. These liquid bridges 51, 52 are formed by the distortion (or crescent shape) of the liquid surface 30 in contact with the edge of the opening 42.

Collector 40 further includes an exhaust conduit 43 connected to a suction device, not shown. This suction device enables the suction of air contained in the collector. This suction causes air to flow through the opening 42, which moves the top of the liquid contained in the chamber via the liquid bridges 51, 52. As a result, the molecular film 30 on the liquid surface is permanently moved by the air circulating in the collector and removed from the liquid surface.

The length L of the opening should be chosen to be at least twice the capillary length K ⁻¹ of the liquid contained in the chamber. This feature forms two side crescent shapes and thus ensures non-sealing of the opening 42 by liquid.

Liquids commonly used to treat cells have capillary lengths between 1 mm and 2 mm. The length L of the opening can thus be preferably 2-4 mm.

The collector 40 is positioned stepwise retracted from the wall by a distance d. In fact, the opening should not be too close to the wall 11, in which case the capillary force forms a single liquid bridge that seals the opening 42 and emptyes the liquid it contains from the lid 20. Because.

However, the opening 42 should not be too far from the wall, in which case the collector cannot have any effect on the liquid.

Furthermore, a distance of a few microns d forms a convex crescent, which has several drawbacks.

First increase the surface tension around the bacterium 10 towards the bottom of the sheath 20, which complicates the operator in locating or removing the cells,

Second, the convex shape deforms the light path of light and makes it difficult for the operator to see the cell.

As a result, it is desirable to maintain the crescent shape in a stable convex shape. For this reason, the distance d is preferably 10-30 microns. Thus, in the collector 40 shown in Figure 2, the distance d is 20 microns.

Preferably, the rims 21, 22, 23 of the chamber walls 11, 12, 13 are preferably covered with a hydrophobic material except the entire surface of the opening 42 of the collector, which is covered with a hydrophilic material. This arrangement facilitates the initiation of the liquid bridge at the collector 40 level.

In FIG. 3, the collector 40 is shown in the form of an end along the discharge conduit 43. The path of inhaled air is shown by the arrow.

Claims (10)

A surface of the liquid contained in the chamber 100, comprising a chamber 100 for containing the cells 10, including at least one liquid injection conduit 121, 122, 123, 124, 125, 126. In the cell culture apparatus configured to be in free contact with the air, It is possible to form at least one liquid bridge 51, 52 with the free surface of the liquid, which is located at the level of the free surface of the liquid contained in the chamber 100 and is maintained at low pressure so as to maintain the liquid bridge 51, 52. And a collector member (40) comprising a mouthpiece (42) for absorbing an air flow to move the surface film (30) of the liquid via the liquid. The collector member 40 is positioned on the rim 21 of the wall 11 of the chamber 100, the mouthpiece 42 of which retracts from the wall 11 in a stepwise manner. Cell culture apparatus, characterized in that located. 3. A cell culture apparatus according to claim 2, wherein a part of the rim (21) located in front of the mouthpiece (42) of the collector member (40) is covered with a hydrophilic material. 4. A cell culture apparatus according to claim 2 or 3, wherein the mouthpiece is positioned stepwise withdrawn from the wall (11) at a distance d of 10-30 microns. The cell culture apparatus according to any one of the preceding claims, characterized in that the mouthpiece (42) of the collector member (40) is elongated and extends longitudinally along the edge of the wall (11). 6. The cell culture apparatus according to claim 5, wherein the opening (42) has a length of 2-4 mm. The cell culture device according to claim 5 or 6, wherein the opening (42) has a height of 0.15-0.30 mm. The cell culture apparatus according to any one of the preceding claims, wherein the mouthpiece (42) is rectangular in shape. The cell culture apparatus according to any one of the preceding claims, comprising a plurality of collector members (40) located along the edges of the walls (11, 12, 13) of the chamber (100). A chamber 100 containing at least one liquid inlet conduit 121, 122, 123, 124, 125, 126, wherein the surface of the liquid contained in the chamber is configured to receive a cell 10 in free contact with air; A method for adjusting the level of liquid included, It is possible to form at least one liquid bridge 51, 52 with the free surface of the liquid, and this flow of air is caused by the suction around the liquid bridge 51, 52 so that this flow of air is the liquid bridge 51, 52. To move the surface film (30) of the liquid via e).