KR100404296B1 - Beating media tissues culture method and beating media tissues culture system - Google Patents

Abstract

The invention relates to a culture vessel (10) connected to a gas supply system (55) to create a culture gas atmosphere, a slide mesh (20) for contacting one surface of a tissue (60) to be cultured in a pulse medium, and a slide mesh. It was configured as a medium nozzle 42 for supplying the medium to the (20), the slide mesh 20 is installed in the culture vessel 10 with the medium composition gradient (21), the medium flow path to the slide mesh (20) A medium flow plate 20d for forming 23 is formed, and a medium flow path 23 for wetting the culture mesh on the medium flow plate 20d and a tissue 60 to be cultured are implanted onto one surface of the tissue. A culture mesh (20u) for contacting the feeder is installed, a medium supply hopper (22) is installed on the medium flow path (23), and the medium provided from the medium supply system (44) is a slide mesh (20) hopper. The discharge nozzle 42 for supplying to the 22 in the pulse flow period to the lid 14 of the container 10. It is a pulse flow medium tissue culture apparatus which mounts and arrange | positions at least one nozzle tip 42a provided with the medium discharge hole 44 to the medium nozzle 42 corresponding to the medium hopper 22. According to the present invention, there is provided a method for culturing a varicose veins medium tissue and an apparatus for culturing a veins of a medium in a biochemical, morphological and mechanical manner.

Description

BREATION MEDIA TISSUES CULTURE METHOD AND BEATING MEDIA TISSUES CULTURE SYSTEM}

The present invention relates to a method for culturing a pulse medium culture tissue and a device for cultivating a pulse culture medium, and more particularly, to provide a tissue culture apparatus having improved biochemical, morphological and mechanical efficiency.

Liver cells cultured by the cell culture method are actively used in in vitro experiments, but the liver cells cultured in the cell state cannot explain the state in vivo in the morphological experiments because the correlation between the cells and the connective tissue is not considered.

The first liver tissue culture was initiated in 1958 with the development of liver tissue culture apparatus and appropriate media by Trowell OA. Experimental Cell Reaseach 16: 118,1959. Trowell's liver tissue culture is a static tissue culture, in which the liver is cut to a suitable size and thickness, placed on a filter paper, and the filter paper is slightly moistened with a cuture medium to allow the tissue to diffuse into the liver tissue. Oxygen required for the metabolism of liver tissue is absorbed from the air through the unattached liver tissue surface. One of the liver tissue culture apparatuses developed after Trowell used stainless steel meshes (meshes) of various sizes instead of spaces. Amino acids and vitamins were added to the culture medium, and the buffer solution was improved in consideration of the osmotic pressure of the medium. Fetal bovineserum (FBS) was used to replenish nutrients in artificial media. However, hepatic cells have high cell metabolism and have been found to have very low cell viability due to insufficient supply of necessary oxygen through the tissue cutting plane. As a result, culturing differentiated adult liver tissue cells remains a particularly difficult task.

Ingram RL.Experimental Cell Reaseach 28: 370,1962, in 1962, reported that 95% O ₂ , 5% CO ₂ gas could increase the survival rate of liver tissue, and Campbell 1971 AK.Experimental Cell Reaseach 68: 33,1971) has shown that survival of liver tissue and liver function (DNA, RNA and glycogen) can be maintained when 95% O ₂ , 5% CO ₂ is supplied at 3atm or less. Reed BB and Grisham JW.Laboratory Investigation 33: 298,1975 reported that in addition to FBS, substances such as insulin and hydrocortisone may be helpful for the culture of liver tissue.

In the tissue culture apparatus commercially available in FIG. 1, the medium is placed in the grid 120 center medium plate 130 of the arm large plate 110 to wet the Millipore filter 140, and the tissue 150 is placed at the center of the filter medium. will be. This culture device is divided into the contact surface between air and tissue and the contact surface between the medium and tissue during tissue culture, and necrosis occurs due to oxygen deficiency in the center of the tissue, and the growth and differentiation activity is increased due to the limited thickness and size of the tissue. ) Was able to culture organs. However, it is not suitable for adult tissues, especially for liver tissue cultures of adults with high metabolism, active material synthesis, and high oxygen demand.

In 1985 to 1986, Smith (PF. Et al. Life Scences 36: 1367, 1985), etc., the rotary tissue culture system (dynamic organ culture system) is a cylindrical mesh (210) attached to the tissue 220 on the inner surface as shown in FIG. ) And the appropriate amount of culture medium 240 into the cylindrical glass drum 230 and sealed, and then supply 95% O ₂ , 5% CO ₂ while rotating the glass drum to repeatedly contact the surface and the medium. I'm trying to. Although this culture apparatus presented many possibilities, the rotation of the cylindrical mesh was not smooth, and the medium exchange cycle was shortened due to the small amount of medium to be put in due to the structural constraints of the glass drum and the part contacting the lowest medium of the cylindrical mesh. Increasing the gas pressure in the culture vessels is known to have high cell viability, which is unsuitable for imparting these conditions. Moreover, the biochemical survival rate of the cultured tissues was verified through this method, but the reliability of morphological survival rate and culture conditions have not been established.

The tissue culture method proposed by Park Seong-su (Patent No. 129833, filed on Nov. 9, 1994) in 1994 is to allow the area and time for the mesh to contact the medium and the air to be 1: 3, respectively. Insert a square column mesh (mesh 200 ~ 400㎛) 330 inscribed in the culture tube into the cylindrical culture tube 31, attaching the tissue 320 to the inner surface of the mesh, and the medium (to contact one side of the mesh) 340) is placed in the culture tube 310, the culture tube 310 is sealed, and while rotating the culture tube at 5 ~ 8 / rpm and 95% O ₂ , 5% CO ₂ is supplied at 3 atm.

In addition, Park's tissue culture apparatus includes a means for supplying sterilized gas to the culture tube at high pressure, and the culture tube uses a Pyrex glass tube to endure at least 3 atm and to autoclave. Sampling cap (sampling cap: 5) is provided, the cylindrical culture tube uses a rotary joint, the gas sterilization system (filter pore size 0.22㎛) in the middle of the gas inlet tube, the temperature of the culture tube in the incubator In a range (37 ± 0.5 ° C for culturing human tissues, 38 ± 0.2 ° C for culturing rats or dogs), and in Smith's open system, gas pressures of 1 to 1.5 atm Pressure can be applied at -3 atm or more, culture medium can be increased, medium need not be replenished frequently, good buffering capacity, It is possible to cultivate 200-500 μm thick tissues larger than 150-300 μm thick, making clear advances in the efficiency of tissue culture.

Specifically, the results of Park's tissue culture method are as follows.

<Organization extraction>

S.D. The rats of the strains were cut heads and livers were removed aseptically, and liver tissue sections having a thickness of 260 µm and a size of 2 mm2 were obtained, and liver tissue culture was performed.

<Condition>

Mesh is mesh size 260㎛

Culture tube is Pyrex glass

The rotation speed of the culture tube is 7 rpm

Used gas 95% O ₂ , 5% CO ₂ Every 10 minutes with 1.7 atmosphere

Culture filter pore size 0.2㎛

Exchange of medium is 1/2 of medium every 12 hours

Incubation time 48 hours

<Culture amount>

Weymouth B752 / 1 Powder Medium (Gibco)

10% fetal bovine serum (B.M.)

2.2% Sodium Bicarbonate (Sigma)

25 mM / ml crystalline bovine insulin zinc (C.R.)

Antibiotic Mixture (Gibco)

<Verification of survival of cultured tissue>

(1) biochemical methods

LDH activity

After culture, 5 minutes, 10 minutes, 30 minutes, 1 hour, and after that, take 100 parts of the culture solution every 2 hours, and measure the LDH activity released into the culture by UV spectrophotometer (Unicam), Protein content was measured by Lowry method and LDH activity was expressed in unit / g protein.

* Fragment ATP amount and DNA amount

Every 2 hours after incubation, 10 cultured tissues were taken, weighed, homogenized, and centrifuged. Then, the supernatant was measured using the ATP measuring kit (Sigma) to measure the fragment APT / g wet weight with spectrophotometer. With the precipitate, the amount of DNA in the cultured liver tissue was measured using DAPI (4 ', 6-diimidino-2-phenylindole).

(2) morphological method

* Optical microscope observation

Every 24 hours after incubation, the culture tissues were taken, fixed in 10% neutral buffered formalin, and paraffin embedded, followed by HE staining, followed by optical microscopic observation. Survival was determined by.

* Electron microscopy

The cultured tissue was taken every 24 hours after incubation, refrigerated and fixed in a mixed solution diluted with 2% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer (pH 7.4) to 70 nm thick. Ultra-thin sections were obtained and counterstained with uranyl acetate and lead citrate, and observed with a Zeol 1200 EX-2 electron microscope, based on criteria such as loss of cytoplasm, destruction of cell membranes, nuclear condensation, loss of chromatin and swelling of individual bodies. Survival was determined.

By virtue of the above criteria, the survival rate of the tissue sections showing the cell survival rate and the cell survival rate of 50% or more in the cultured tissue was divided and the respective survival rate was calculated by the following equation.

* Survival rate of cultured tissue

Staining tissues cultured for 48 hours with HE under light microscopy showed distinct nucleolus and cytoplasm basophils in surviving cells, and severe nuclear condensation and cytoplasm staining eosinophilic in dead cells. Could. HE staining of the longitudinal and transverse sections of the cultured tissue showed no central necrosis and the entire layers of gas-tissue junction and culture-tissue junction surface survived. When observed under electron microscope, severe nuclear condensation or loss of chromatin, loss of cell sol, damage of cell membrane, and severe edema of private bodies were observed in dead cells. Life and death judgments were possible.

As shown in Table 1, the survival rate (area) by morphological observation was 76.0 ± 11.4% on average, and the survival rate (tissue section) was excellent by 71.5 ± 5.0% on average.

TABLE 1

Group Ⅰ Ⅱ Ⅲ

Survival rate (area) 74.1 ± 12.9 79.6 ± 9.3 77.2 ± 8.8

Survival Rate (Tissue Section) 73.8 67.7 67.7

* Biochemical Survival Verification

LDH activity released from the tissue into the culture medium over time was 170.8 ± 6.14 at 5 minutes, 186.42 ± 6.72 at 10 minutes, 203.27 ± 5.03 at 30 minutes, and 210.66 ± 14.25 units / g at 1 hour. Thereafter, LDH activity measured at 3 hour intervals did not show a significant change of around 200. On the other hand, the control group left at room temperature after 6 hours incubation showed a continuous increase in LDH activity.

The amount of ATP and DNA in the pre-culture and the tissue up to 48 hours after incubation remained almost insignificant.

As described above, there was no significant difference between the cultivated by the method and the apparatus according to the composition of the Park Sung-soo and the morphologically uncultured tissue, the damaged gas-tissue and culture-tissue when the section is made from the section of the culture tissue Except for one or two cell layers present at the junction, hepatic cells in most cell layers survived well.

However, Park Sung-Soo's culture apparatus has the following improvements despite the achievements.

First, by implanting the tissue inside the square pillar mesh and rotating the culture tube to wet the mesh, the medium supply cannot be precisely controlled.

Second, since the mesh is to use the inner surface of the mesh of the square pillar shape is difficult to think of the tissue.

Third, implantation of tissue on a moving mesh results in unstable culture tissue management.

Such mechanical problems show that Park's tissue culture device is a device that cannot strictly carry out its culture method.

Specifically, Park Sung-Soo's culture apparatus is a tissue to be cultured in a cylindrical culture tube maintained in a 95% O ₂ , 5% CO ₂ , 3atm gas environment in order to maintain the area and time that the tissue is in contact with the medium and air at 1: 3 respectively. After inserting the square pillar mesh (mesh 200 ~ 400㎛) that is implanted, the culture tube is rotated to 5 ~ 8 / rpm. As a result, one side of the square pillar mesh is wetted with the medium in the culture tube. Such a rotary tissue culture device is in an unstable state by implanting the tissue on a rotating body, and by rotating the culture tube at a uniform speed, the time and area of contacting the tissue with the medium and the air are 1: 3, respectively. It is difficult, and more difficult to control the contact time tube area at different ratios.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for culturing a varicose veins medium tissue and an apparatus for culturing veins of a vein medium directed toward a metabolic environment of a living body.

Another object of the present invention is to provide a method and apparatus for culturing varicose vein medium that can be widely used in experimental space, surgical space, vaccine development, drug development, and the like.

Another object of the present invention is to provide a varicose vein tissue culture apparatus that can be easily used and precisely control the culture conditions.

Figure 1 (A), (B), (C) is a commercial tissue culture apparatus

Figure 2 (A), (B), (C) is Smith rotary tissue culture method

Figure 3 Park Sung-soo (Patent No. 129833) culture apparatus

4 is a flowchart of a medium circulation system of a conventional tissue culture system.

5 is a perspective view of this invention mesh

Fig. 6 (A) (B) is a cross-sectional view and an enlarged cross-sectional view of the present invention mesh embodiment, and (C) (D) is a cross-sectional view and an enlarged cross-sectional view of another embodiment of the present invention mesh.

Figure 7 is a perspective view of an embodiment of this invention nozzle

8 is a perspective view of an embodiment of the open state of the culture vessel of the present invention

9 is a perspective view of another embodiment of the culture vessel of the present invention

Figure 10 is a cross-sectional view of the use state showing the invention medium circulation system

Figure 11 is a cross-sectional view of the use state showing the invention gas circulation system

<Description of Drawing Major Symbols>

20: mesh 21: tilt 22: hopper 30: cassette 40: medium feeder 42: nozzle 42a: nozzle tip 50: circulation feeder 51: gas flow path 53: gas supply horn 54: gas suction drain

4 shows a medium circulation system of a conventional space- or tissue culture apparatus.

The culture medium is a recovery tank (SE), a separator (Se), a secondary tank (Su), a storage tank (ST1), a pump (Pu1), one or more culture vessels (CS), a storage tank (ST2), a pump ( Proper contact with the culture tissue in the culture vessel CS is continued in the metabolism of the culture tissue while circulating the medium circulation system leading to the PU2), the storage tank ST3 and the pump PU3. To this end, the culture vessel CS connects the culture gas circulation system in addition to the apparatus of FIG. 4 to create a 95% O ₂ , 5% CO ₂ gas atmosphere for respiration of the tissue in the culture vessel CS.

The devices forming the system of FIG. 4 can be added or subtracted as needed.

When using a tissue incubator as an experimental or research in-spatial system, the liver of the experimental animal is removed and the liver of the experimental animal is replaced as the culture vessel (CS). In this case, the liver is cultured by the heartbeat of the test animal's blood and heart, and the cultured tissue and blood can be observed and tested in the culture vessel (CS).

The culture apparatus of the present invention will be described in detail with reference to the accompanying drawings as follows.

In the present invention, the culture gas means a culture gas required for tissue culture based on 95% O ₂ , 5% CO ₂ gas.

In the present invention, the contact between the medium to be cultured and the medium to be cultured under the culture gas atmosphere was set as follows. .

The slide mesh 20 is installed in the culture vessel 10 with the medium flow composition gradient 21, the medium is supplied to the hopper 22 of the slide mesh 20 at a pulse flow cycle, and the pulse flow vessel formed in the medium flow path 23 is formed. Wet the culture mesh (20u) as a tributary, and the tissue 60 is cultured by implanting the tissue 60 to be cultured so that one surface of the tissue (60) in contact with the pulse medium medium onto the culture mesh (20u).

The drainage medium culture method of this invention will be further clarified by the pulse medium tissue culture apparatus of the examples below.

The tissue culture apparatus includes a slide mesh 20 for connecting a culture vessel 10 connected to the gas supply system 55 to form a culture gas atmosphere, one surface of the tissue 60 to be cultured in the pulse medium, and a slide. It is comprised as the medium nozzle 42 for supplying a medium to the mesh 20.

The slide mesh 20 for tissue implantation is installed in the culture vessel 10 with the medium composition gradient 21. A medium flow plate 20d for forming the pulse flow medium flow path 23 in the slide mesh 20 is configured. On top of the medium flow plate 20d, the culture medium 23 for wetting the culture mesh and the tissue 60 to be cultured are implanted to install a culture mesh 20u for contacting the medium on one surface of the tissue. The hopper 22 for medium supply is installed in the upper part of the medium flow path 23. A medium nozzle 42 for supplying the medium supplied from the medium supply system 44 to the slide mesh 20 hopper 22 in a pulse flow period is mounted on the cover 14 of the container 10. One or more nozzle tips 42a having the discharge discharge holes 44 formed in the discharge nozzle 42 are arranged in correspondence with the arrangement of the discharge hopper 22.

The culture vessel 10 is a rectangular culture vessel of Figure 8, a cylindrical culture vessel of Figure 9 or an oval culture vessel. Cylindrical culture vessels are advantageous when the pressure of the culture gas is high, but an elliptical or rectangular pressure vessel may be used to increase the array density of the mesh in the culture vessel. In the embodiment of the present invention, the density of the slide mesh 20 was increased by selecting a rectangular pressure vessel capable of withstanding internal pressure of 1 kg / cm 2 or more. Specifically, the opening and closing cover 12 is mounted with the seal 11 for maintaining airtightness on the culture vessel 10. The cover 12 is assembled to the container 10 as a hinge 13, and presses the cover 12 to maintain the internal pressure of 1kg / ㎠ or more as the fastener 12.

In the embodiment, the culture vessel 10 is composed of a transparent wall 10a, an upper flange-type metal material 10b, and a lower drain-type bottom 10c, and a transparent upper plate 12a in the center of the cover 12. ) Was placed.

The size of the slide mesh was 50 mm long and 150 mm long in the embodiment. There is no particular meaning to this size, but the quantity of tissue 60 to be implanted and implanted within the container is considered. In particular, when using the incubator as a space, it is necessary to consider that the slide mesh should be installed at a capacity that can replace the role of the animals.

In the embodiment, the culture medium composition gradient 21 of the slide mesh 20 is set to ∠40 to 80 °, the height of the medium flow path 23 is 0.8 to 1.5 mm, and the culture mesh 20u of the slide mesh 20 is used. The mesh wood 200 ~ 400㎛ using a stainless steel mesh (20g) or non-toxic synthetic resin, medium flow plate (20d) and the hopper 22 is a stainless steel plate or non-toxic synthetic resin, respectively, the slide mesh 20 medium flow Sliders 20s were formed on both sides of the plate 20d, and both sides of the culture mesh 20u were attached to the sliders 20s.

The height of the medium composition slope 21 and the medium flow path 23 is set so that the medium 70a supplied to the hopper 22 flows through the medium flow path 23 as the medium 70b and evenly wets the culture mesh 20u. The feeder is adjusted to have the flow rate of the selected medium.

6 (A), (B), (C), and (D) show that the height of the medium flow passage 23 is 0.8 to 1.5 mm in order to increase the installation density of the slide mesh 20 in the container 10. 75 degrees. In particular, FIGS. 6 (C) and 6 (D) form a cover mesh 24 detachable on the culture mesh 20u so that the tissue 60 implanted on the culture mesh 20u does not escape from the culture mesh 20u. Doing.

The medium flowing in the medium flow path 23 may be compared to blood flowing in the blood vessels of the experimental animal in the present invention. The flow rate and flow rate of the medium take into account the relationship between the tissue and the blood vessels in the experimental animal. In addition, in order to bring the medium 70b closer to the blood in the blood vessel, the medium supplied to the hopper 22 is interrupted in a constant pulse flow cycle. In this case, the medium that wets the culture mesh 29u forms a varicose medium, and the cultured tissue implanted in the culture mesh 20u is in contact with the varicose medium to metabolize.

In this invention, in order to pulsate the medium, the driving of the medium supply pump is paused at any necessary period, and the RPM of the medium supply pump is controlled to adjust the medium supply amount.

The pulse medium of the present invention acts as a change in osmotic pressure on the tissue 60 implanted on the mesh 20, the change in osmotic pressure to the tissue is understood as a pumping action to promote the metabolism of the tissue. The controllable pulse flow cycle is the biochemical cycle of the tissue (eg pulse cycle), and the setting of the pulse flow cycle and the pulse flow characteristics for a specific tissue is the subject of new experiments and studies.

In order to maintain the inclination of the slide mesh 20, a slide mesh 20 supporting means is placed in the culture vessel 10. As the mesh support means, the cassettes 30 of FIGS. 10 and 11 are provided with mesh supports 35 and 35a which are housed in the container 10 and allow one or more meshes to be arranged continuously.

In the present invention, the atmosphere gas composition device applied to the culture vessel 10 is provided with a horn 53 for supplying the atmosphere gas to one side of the atmosphere gas movement route 51 in the culture vessel 10, and the atmosphere gas movement route 51. At the end, a drain 54 for inhaling the atmosphere gas was installed.

The medium feeder 40 of the present invention receives the medium from the medium supply system 44 including the medium transfer pump 45, and forms the medium in the form of water droplets 70 on the slide mesh 20 mounted in the culture vessel 10. The medium passing through the slide mesh 20 is recovered to the medium supply system 44 through the drain 15 at the bottom of the culture vessel 10.

The medium supply system 44 depends on its use, such as when using a tissue culture apparatus as an experimental in-patient, when it is used for the development of medicines such as vaccines, etc., but the example of Figure 4 applies mutatis mutandis.

In the present invention, the medium feeder 40 is a device dropwise dropwise in the form of water droplets 70 into the hopper 22 of the mesh 20 through the nozzle 42 through the medium fed by the pump 45. The nozzle is connected to the medium supply hose 43 and supported by the container 10 to drop the medium onto the hopper 22 of the mesh 20 in the container 10. The addition amount adjustment of the medium is made by controlling the driving speed (RPM) of the pump 43 in consideration of the discharge capacity of the nozzle 42. In the embodiment, the diameter of the medium supply hole 44 of the nozzle tip 42a is 0.3 to 1 mm, specifically 0.4 mm, in order to drop the medium used in the conventional experiment in a pulsating period, and the pump so that the medium forms water droplets from the hole. The medium was added dropwise by adjusting the supply pressure of the medium. To drop the medium onto one or more meshes, one or more tips 42a are arranged in the nozzle. In this embodiment, a tree-type nozzle in which 14 nozzle tips 42a are arranged in a tree type corresponding to the mesh hopper 22 arrangement is constructed, and two tree-type nozzles are installed in one culture vessel cover 12. It was accommodated in 28 mesh in the culture vessel of to enable tissue culture in 28 mesh at one time.

In order to control the medium supply amount, the medium droplet 70 emitted from the medium dropping nozzle is detected as an optical sensor (not shown), and the medium droplet is fed back to the pump control unit (not shown) to control the medium supply amount, and the medium supply is performed. By intermittently driving the pump 43, the pulsating medium can be formed in the medium supply slide mesh 20.

The culture gas supply unit 50 receives gas from the gas supply system 55 including 95% O ₂ , 5% CO ₂ storage tank, auxiliary tank, sterilization tank, filter and pump, and the like into the culture vessel 10. The supply and the culture gas used in the vessel 10 to recover the gas in the supply system (55).

In FIG. 11, the gas provided by the gas supply system 55 is the gas of the mesh yard inside the container 10 through the port 53a installed in the cover 12 and the supply horn 53 installed in the container 10. The culture gas is supplied to the movement path 51, and the culture gas passed through the path 51 is recovered to the gas supply system 55 through the suction drain 54 and the port 54a. Selection is made according to the supply pressure of the culture gas and the conditions for circulating the culture gas.

As described above, the present invention provides a method for culturing a pulsed medium tissue and an apparatus for culturing a pulsed medium tissue directed toward a metabolic environment of a living body, and can be widely used for laboratory space, surgical space, vaccine development, and drug development. It is to provide a tissue culture method and apparatus, the medium nozzle is installed on the cover to open the culture vessel, the slide mesh can be easily accessed, while the culture tissue can be handled by the slide mesh unit, so that the biochemical, morphological and mechanical It is to provide a tissue culture apparatus with improved efficiency.

Due to these achievements, the present invention is expected to be substituted for experimental and surgical citations and widely used in vaccine development and drug development.

Claims (3)

(Correction) Pulsating the medium to the slide mesh by incubating the tissue in contact with the medium under a 95% O ₂ , 5% CO ₂ culture gas atmosphere, and installing the tissue culture mesh with the gradient of the medium composition in the culture vessel. A pulse culture medium culture method characterized by inducing a pulse medium to the culture mesh by dropping in a cycle, and implanting the tissue to be cultured on the culture mesh to contact with the pulse culture medium. (Correction) A culture vessel that creates a 95% O ₂ , 5% CO ₂ culture gas atmosphere, a mesh installed at a constant slope in the culture vessel so as to contact one surface of the tissue in the culture vessel, and a slide mesh What is claimed is: 1. A medium flow supply plate configured as a medium flow supply nozzle for guiding a feeder, comprising: a medium flow plate disposed proximate to a bottom surface of a slide mesh to form a medium flow path; Pulsed medium tissue culture apparatus characterized in that it comprises a nozzle for dropping the medium in the upper pulsation period in the medium passage in order to induce the pulsating medium in the culture mesh. (Correction) The medium according to claim 2, wherein the medium composition gradient of the slide mesh is? 40 to 80 degrees; The height of the medium passage is 0.8-1.5 mm; Mounting a medium nozzle on the lid of the culture vessel; An atmosphere gas supply horn is installed on one side of the atmosphere gas movement path in the culture vessel; An atmospheric gas intake drain is installed at an end of the atmospheric gas flow path; A varicose medium tissue culture apparatus characterized by installing a drain for medium recovery at the bottom of the culture vessel.