JPH043193B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a cell suspension and transferring it to continued operation.

Methods of this type are for producing cell suspensions, for example suspensions of yeasts, bacteria, cultured tissues, microorganisms, etc., from original cell populations. The cell suspension is transferred to a mounting plate or to an incubator with synthetic growth medium in order to isolate mutant strains and, more generally, to study cell traits.

A well-known method for transferring cells is the replica method, which was developed by Lederberg in the United States in 1952 and subsequently became popular. There, a large number of original cell colonies are grown in an incubator, then pressed onto velvet, and from there copied onto another flat plate. In this method, the properties of the cells on the first plate are changed to the properties of the cells on the second plate by changing the components of the medium on the second plate or by physically or chemically treating the cells on the second plate. It can be investigated by comparing.

However, the disadvantage of this method, which is used worldwide today, is that the size of the cell colonies obtained from the first plate is often variable, resulting in a highly variable number of cell colonies on the second plate. . Furthermore, the thickness of the cell membrane on the second plate can also vary widely, which makes physical tests of cell properties, such as UV irradiation and chemical tests, difficult, and the measurement results can vary due to varying light transmittances, for example. makes it inaccurate. A disadvantage of this method can also be seen in the inability to produce cell suspensions, which are sometimes absolutely necessary for characterizing and processing cells.

The aim of the present invention is to provide a method by which cell suspensions can be reliably created on individual cell colonies. The purpose of this is to: - Cultivate a colony of protocells in a culture vessel containing a growth medium, - Transfer cells linearly from the colony to the culture vessel using a blade-shaped transport device having multiple blades extending parallel to each other. - After a predetermined culture period, the blade-shaped transport device is pressed against the culture vessel by crossing the first replica cells, → column-shaped cell colonies that have grown at predetermined intervals during the culture period. - After a predetermined culture period, the cells are removed from the cell and transferred to another culture vessel (on-point stacking) by a pin transport device with movable pins at intervals corresponding to the spacing of the blades of the blade-shaped transport device. , remove cells from individual cell colonies grown during the culture period, transfer them to a multi-sample support having a large number of wells, prefilling the wells with a solvent, - shaking the support, - this The cell suspension generated in the support by shaking is transferred to the treatment culture vessel by a round-head transport device with a number of round-head pins.

This is achieved through the above steps. These measures provide a method by which individual cell colonies can first be cultivated and cell suspensions made from these individual cell colonies for further processing.

In a variant of this method, - a nearly turf-like cell colony (cell turf) is cultivated; - cells are removed from the cell turf by a fine pin transport device having a large number of fine movable pins, and Transfer it to a well containing a growth medium, while filling the well of the multi-sample support with the same amount of liquid growth medium as the number of cells required for culture; - After a predetermined culture period, transfer the multi-sample support to the well. The cell suspension produced in the recess is taken out from the recess of the multi-sample support using a round-head transport device having a large number of movable round-head pins, and transferred to a processing incubator for continued operation. scheduled. In this method, a thin pin transport device is used instead of a blade transport device.
This fine pin transport device has fine pins with a diameter of 0.01 to 0.1 mm. This small pin diameter ensures that cells are removed from the original cell colony.

To configure this embodiment, the multi-sample support is shaken to promote cell growth after cell addition;
Plan to dilute the cell suspension with buffer before transferring to the treatment incubator. The cell suspension produced in this way is thus obtained directly in the wells of the multimaterial support, and the growth medium is in such a volume that it is used up after the end of the culture period. Metabolites and nutrients that cannot be digested by the cells to be suspended in each case remain in solution. Generally, these impurities are not a hindrance, and even if they are, they can be neutralized with a buffer.

The invention further aims to provide a device capable of transporting limited quantities of cells or cell suspensions. This objective is achieved by a support plate in which a support pin is movably supported in a bore. To construct this embodiment, the hole extends approximately at right angles to the plane of the support plate, and the support pin is free to move within the hole and is held by its own weight to the support plate by a retaining collar. In order to always ensure the same penetration depth even with different thicknesses of cell colonies to be transferred, the outer dimensions of the retaining collar are larger than the inner dimensions of the holes, and the retaining collar is designed to allow individual adjustment of the mass of the support pins in the support plate. The size of the colors is individually variable. These measures ensure that the support pin always penetrates the same depth into the cell colony and removes approximately the same amount of cells from it.

In order to transfer the cell suspension to an incubator for continued processing, the support pin has a round head at the end remote from the support plate, and the outer dimensions of the round head are smaller than the shaft of the support pin with the round head. big. These measures result in a transport device with a large enough surface to remove the amount of cell suspension required for continued processing from the recesses of the multi-sample support and transfer it to the incubator. The spacing between the support pins or the spacing between the round head pins is matched to the spacing between the recesses of the multi-sample support.

In order to make the penetration depth of the round head pin uniform, the support pin is held by a retaining ring placed on the opposite end of the shaft from the round head, and the retaining ring of the support pin is a removable plastic ring. It is advantageous if By means of these devices, the round-head pin is held elastically and easily and freely movable within the support plate.

A device consisting of a plurality of blades held approximately parallel to one another and spaced apart by spacers is used to transfer a limited number of microbial cells from a cell colony to an incubator and then distribute them into individual cell colonies. . The blades are therefore held equidistant from each other by spacers, which spacing is equal to the spacing between the support pins in the support plate. The blades extend approximately perpendicular to the axis of the spacer to reduce the risk of adjacent cell colonies interacting with each other or intersecting at the interface.

In order to use the method described above according to the invention, this method is used for the production of a cell suspension and this cell suspension is used for the isolation of mutant strains. In order to use the device described above according to the invention, this device is used for the production of a cell suspension and this cell suspension is used for the isolation of mutant strains.

The present invention is illustrated in the drawings and will be described in detail below.

The pin transport device 10 shown in FIG.
It is provided in A support pin 12 is inserted into this hole 13 and held. The support pin 12 has play and is easy to move, but is guided so as not to tilt. Support plate 1
1 , the support pin 12 is held by a retaining collar 14 and hangs freely in the hole 13 under its own weight. In order to easily insert the support pin 12 into the hole 13, the support pin has a protrusion 15.
has. The protrusion 15 and the retaining collar 14 can be used to adjust the mass of the support pin.

With this type of pin transport device 10, limited amounts of cells can be removed even from original cell colonies of different thicknesses. Since the support pins 12 have the same mass, the depth at which they penetrate into the cell colony is uniform even when the growth height of the cell colony is different. The removed cells are transferred to a sample support 16 as shown in FIG. The sample support 16 has approximately the same planar dimensions as the support plate 11 and has a recess 17 at a position corresponding to the position of the support pin 12.

In the embodiment shown in FIG.
2 has a rivet-like round head 18 at the end remote from the support plate 11. This round head has an undercut 31 relative to the pin shaft 29. A retaining ring 19 holds this support pin 12. The retaining ring is support plate 1
It is fitted onto the free end of the support pin on the side opposite to the round head 18 of 1. The retaining ring 19 is made of a resilient material, for example plastic, and is secured with a sturdy seat.

Figures 4 and 5 show a large number of mutually parallel blades 2.
This is a blade-shaped transport device 20 consisting of 1. The blades 21 are held at uniform spacing 34 by spacers 22 and connected by retaining means 23, for example rivets or nuts.

With this type of blade-shaped transport device 20, it is possible to copy the vertically extending copy blade shape 25 of the cells to be transferred onto the culture vessel. By applying various growth media to the culture vessel 24, vertically extending cell colonies 27 can be generated. To isolate a mutant strain or obtain a punctate cell colony, the blade-shaped transport device 20 is once again pressed across the row-shaped cell colony 27 to form a laterally extending replica blade as shown in FIG. Shape 26 is replicated onto another media support.

Since this incubator 32 also has a growth medium, the cells carried away by the blade-shaped transport device 20 when intersecting the row-shaped cell colony 27 are transferred to the new dot-shaped cell colony 2.
8 can be generated. This punctate cell colony 28 serves to separate the mutant strain, which can be received by the pin transport device 10 and transferred to the sample support 16. Punctate cell colonies 28 often grow irregularly, which can be caused by different growth media. An unoccupied intersection point 33 between the replica blade shapes 25 and 26 indicates that the cells pressed there have specific properties relative to the growth medium or test medium applied to the incubator 32. show. For inspection, this laterally extending replica blade 26 can also be pressed onto another incubator 24 with the same growth medium as the incubator for the arrayed cell colonies 27 .

The device described above can be configured in the same manner as another device (not shown) to simplify the operation.

By the proposed method and using the proposed device, individual cell colonies are arranged at predetermined mutual intervals,
Making a cell suspension from it can be done easily and reliably. The property of cell colonies, which grow faster when there are no other colonies nearby, can be exploited for optimal results.

[Brief explanation of drawings]

FIG. 1 is a sectional view of a support plate and a support pin inserted into it. FIG. 2 is a cross-sectional view of a multi-sample support receiving cells transported by the device of FIG. FIG. 3 is a sectional view of a round head transport device for transferring a cell suspension to the sample support of FIG. 2. FIG. 4 is a front view of the blade-shaped transport device.
FIG. 5 is a plan view of the apparatus shown in FIG. 4. FIG. 6 shows the first replica blade shape obtained by replicating the device shown in FIG. FIG. 7 shows a second replica blade shape that is a replica of the device in FIG. 4, intersecting the replica blade shape that is a replica of the culture vessel in FIG. 6, and a dotted cell colony that has grown on it. 10... Transportation device, 11... Support plate, 12...
Support pin, 13...hole, 14...retention collar, 1
5... Protrusion, 16... Sample support, 17... Recess, 18... Round head, 19... Holding ring, 20... Blade-shaped transport device, 21... Blade, 22... Spacer,
23... Holding means, 24... Incubator, 25... Column copying blade shape, 26... Lateral copying blade shape, 27...
Row-shaped cell colony, 28...Punctate cell colony, 29...
Pin shaft portion, 30...upper surface, 31...undercut, 32...incubator, 33...unoccupied intersection, 34...interval.

Claims (1)

[Claims] 1. In a method for producing a cell suspension and transferring it to continuous operation, an original cell colony is cultured in an incubator containing a growth medium, and a plurality of cells extending parallel to each other are cultured in a culture vessel containing a growth medium. Cells are copied in a line from the original cell colony to the culture vessel using a blade-shaped transport device having a blade (on-lines stamping), and after a predetermined culture period, the blade-shaped transport device is crossed over the first replicated cells. Cells are copied onto a culture vessel, and cells are removed from the linear cell colony that has grown at predetermined intervals during the culture period and copied onto another culture vessel (on-point stamping), and after the predetermined culture period, a blade-shaped transport device is used. A pin transport device with movable pins at intervals corresponding to the spacing of the blades removes the cells from the individual cell colonies grown during the culture period and transfers them to a multi-sample support having a large number of recesses, during which the recesses are preliminarily placed. A solvent is placed in the sample support, the sample support is shaken, and the cell suspension generated within the support by this shaking is transferred to a processing incubator using a round-head transport device having a large number of round-head pins. A method for limited transport of characterized cells. 2. The method for limited cell transport according to claim 1, wherein the processing culture vessel to which the cell suspension is transferred using a round head transport device is a culture vessel having a mounting plate or a synthetic growth medium. 3. In the method of producing a cell suspension and transferring it to continuous operation, a nearly turf-like cell colony (cell turf) is cultured, and cells are removed from the cell turf using a pin transport device having a large number of fine movable pins. , transfer it to the recess containing the liquid growth medium of the multi-sample support, and at that time, fill the recess of the multi-sample support with the same amount of liquid growth medium as the cells required for culture, and after the predetermined culture period. The cell suspension formed in the recesses of the multi-sample support is removed from the recesses of the multi-sample support by a round-head transport device having a number of movable round-head pins and transferred to an incubator for continued operation. A method for limited cell transport characterized by the above steps. 4. The method for limited cell transport according to claim 3, characterized in that the multi-sample support is shaken to promote cell growth after adding the cells. 5. A method for limited cell transport according to claim 3 or 4, characterized in that the cell suspension is diluted with a buffer before being transferred to a culture vessel. 6. The method for limited cell transport according to claim 3, wherein the culture vessel to which the cell suspension is transferred using the round head transport device is a culture vessel having a mounting plate or a synthetic growth medium. 7 A device for transporting cells to extract cells from a cell colony, in which a hole 13 is formed in a support plate 11, and a support pin 12 is movable in the longitudinal direction of the support pin 12 within the hole 13. A device for limited transport of cells, characterized in that the device is supported. 8. The device for limited transport of cells according to claim 7, characterized in that the holes 13 extend approximately at right angles to the plane of the support plate. 9. The device according to claim 7 or 8, characterized in that the support pin 12 can freely move within the hole 13 and is held on the support plate 11 by the support collar 14 due to its own weight. A device for limited transport of cells. 10 A patent claim characterized in that the outer dimensions of the retaining collars 14 are larger than the inner dimensions of the holes 13, and the sizes of the retaining collars are individually variable in order to individually adjust the mass of the support pins 12 in the support plate 11. range 7th
A device for limited transport of cells according to any one of Items 1 to 9. 11. The device for limited cell transport according to any one of claims 7 to 10, characterized in that the support pin 12 has a round head 18 on the side remote from the support plate 11. 12. The device for limited cell transport according to claim 11, wherein the outer dimension of the round head is larger than the shaft portion 29 having the round head of the support pin 12. 13. Claim 11 or 1, characterized in that the support pin 12 is held by a holding ring 19 placed on the end of the shaft portion 29 opposite to the round head 18.
A device for limited transport of cells according to item 2. 14. The device for limited cell transport according to claim 13, wherein the holding ring 19 of the support pin 12 is a removable plastic ring. 15 A blade-shaped transport device having a plurality of blades 21 held apart from each other substantially parallel to each other by spacers 22, and a hole 13 formed in the support plate 11.
A device for limited transport of cells characterized in that the support pin 12 is constituted by a pin transport device movably supported in the longitudinal direction of the support pin 12. 16. A device for limited transport of cells according to claim 15, characterized in that the blades 21 are held at equal intervals 34 from each other by spacers 22, and this interval 34 corresponds to the interval between the supporting pins 12 arranged side by side. . 17. The device for limited cell transport according to claim 15 or 16, characterized in that the blade 21 is provided extending substantially perpendicularly to the axis of the spacer 22.