JPH02501264A - Modification of viable cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Modification of viable cells The present invention provides methods for introducing or fusing substances into living mammalian cells. Concerning the use of ultrasound for medical purposes. Although this method traumatizes cells, these will not die or disintegrate. Ultrasound is generally too expensive to detect with the ear. accompanied by mechanical vibrations at high frequencies, typically 18 kHz to 20 MEt. .

Ultrasound is used for diagnostic and therapeutic purposes on living mammalian tissue. harm Regarding the maximum strength that can be applied without giving Report on Biomedical Engineering, January 1974, pp. 48-51). ing.

Ultrasound can perform complex chemical processes such as breaking down or disintegrating polymers, including DNA. It is widely used to An ultrasonic diswitch grater is available for this purpose. It's on sale.

Scanning ultrasound microscopy utilizes low intensity vibrations in the ME2 to GH range.

There are a wide variety of methods for introducing substances into living cells, including calcium phosphate. precipitation method and electroporation ) involves exposing cells to a Pärnu electric field. It is presumed that this leads to the formation of pores in the plasma membrane. This converts DNA into plants. It has been used to introduce into cells of both animals and animals, and it is difficult to achieve this by other methods. has been effectively applied to a wide range of cell types (G, Cks et al. tVsclaic Ac1ds Re5earch, Volume15 nu mber 3, 1987, p. 1311-1326).

Similarly, there are many different ways to fuse cells, including viruses, Natural methods using e.g. Sendai or HIV, and polyethylene glycol Le-mediated fusion or electrofusion (m1act-rof%hanishiki) This includes artificial methods that involve The electrofusion method consists of two main steps. Ru. dielectrophoresis (diglgctrop), which brings cells into close contact h-orgsia) and create pores in the cell membrane, resulting in close contact. It is an electrical membrane breakdown that fuses two juxtaposed membranes (2.Oniji (K, Oh*1ahi), Josrxalof ImmunologicaL Me thods, 100 (1987) 181-189).

Ultrasonic power can be used to achieve the desired close contact between cells. Ru. Much shorter ultrasound wavelengths are employed than in the case of fusion chambers. This is it This not only results in perfectly spherical chain cells (in purely propagating sound waves), but also in constant At the maximum pressure of the standing wave, cells become concentrated. W, M, Arnold (W, M, A7nold) et al., (Biochats+1cal 5ociety Tra nsactions, 1986, to concentrate red blood cells or myeloma cells. For this purpose, 1.0 Jllh ultrasonic waves (wavelength 1 xi) were used.

The present invention provides for traumatizing the cells in a container, in the presence of a substance, or just prior to the addition of a substance. The substance can be prepared from a living mammal by subjecting it to an ultrasonic excitation treatment sufficient to give A method for introducing a substance into a cell or fusing a substance with the cell is provided.

In summary, cells in suspension are exposed to ultrasound at frequencies ranging from kHz to MEt. expose. These frequencies induce cell vibrations or create cavities near the cells. tion. The stress generated in the cell membrane causes total rupture of the cell and pores in the cell membrane. formation, or fusion of two or more adjacent cells. This cell rupture stage During this period, substances in the solution in which the cells are suspended may be taken up by these cells. There is.

The cells are subjected to an ultrasonic excitation treatment sufficient to traumatize them. This is a thin The vesicle is altered and temporarily damaged enough to allow the apposed material to enter. meaning that it does not die or disintegrate. Possibly, short-term pores form in the cell membrane. , which allows the juxtaposed substances to enter or fuse with the cells. It will be possible. Generate ultrasound waves strong enough to traumatize cells It is inevitable that some cells will die.

The nature of mammalian cells is inconclusive. Cells are preferably suspended in water or other fluid It may be held in a state where it is attached to a support, or it may be processed in a state where it is attached to a support.

The intensity of ultrasound is sufficient to traumatize cells, but not permanently damage them. or are selected to avoid extinction. Appropriate strength depends on many factors and depends on individual practice. It is easily determined empirically for experimental designs. The ultrasound frequency is generally 18 kHz. and 20 MHg. Processing times are chosen empirically and are not acceptable. The temperature rises to such an extent that it stays warm. A total processing time of a few seconds to a few minutes is appropriate. It seems that it is urgent.

Substances taken up by living cells using this method include proteins, nucleic acids, oligonucleotides, Includes DNA, lipids, and lipid vesicles. This during the ultrasonic excitation treatment period Cells that have taken up these substances can survive the treatment and subsequently replicate. this method Genes introduced into cells can be expressed and transmitted to daughter cells in a hereditary manner.

Instead of being introduced into the cell, substances can be incorporated into the cell membrane in this way. . Examples of substances for this purpose include lipids, hydrophobic proteins, membrane receptors, and lipid bases. Includes cells, and ribosomes. Furthermore, using ultrasonic excitation treatment, two Induce fusion of more than one apposed homologous or heterologous cells to form a single cell You can also do that.

Preferably, the cells' are maintained in suspension in water or other liquid medium. Thin The cell concentration is generally between 10' and 108 cells/-. The substance is preferably treated with ultrasonic excitation present, but sometimes during or after the excitation process, the cell may be traumatized. It can also be introduced while the system is still in its current state. Preferably, the concentration of the other substance is also maintained at a high level. should be held.

As discussed below, in order to achieve high local concentrations of both cells and other substances in suspension, Ultrasound and other methods can be used for this purpose.

In the prior art electroporation and electrofusion methods Various known means for improving efficiency are also useful in the present invention. gluco /CaC1,7M gCl, polyethylene glycol, albumin, calcium Lumodeulin, Phosphatidylserine, Glyceryl Monooleate, Cholesterol Adjuvants containing polymers may be present for cell fusion. Suspension pH 1 salt Concentration and temperature are all factors that affect efficiency. Transfer of DNA into cells For transfection, carrier DNA, e.g. The presence of sperm DNA may increase efficiency.

The cells are retained within the container. Its dimensions and shape and materials of construction (acoustic impedance) -dance) must be selected in relation to the ultrasonic processing equipment used. small The shop's (6sjos) polystyrene vial or Alchiwell type can be used with several ultrasonic waves. Can be applied by transducers, these placed on the top, side or bottom of the container or even with a probe that is immersed in suspension in a container good. Alternatively, the suspension may be passed through one or more ultrasonic transducers to a controlled It may be fluidized in a fashion.

Ultrasound or other means (e.g., constant wave).

One ultrasound transducer is installed to concentrate the cells, while the other one concentrates on them. Manipulated to cause trauma periodically. or use the same transducer for both purposes. It can also be used to periodically switch the power setting from a low level to a high level. Hyper Controlling the frequency or frequency spectrum of sound waves allows for cell fusion or material uptake. The loading can be optimized.

The following examples illustrate the invention.

Polystyrene bijos’ vial 9cm Petri dish for tissue culture cell 63°, 4g 8.653 mice suspended in DMEMK containing 10% calf serum Sumieloma cells. DMEM is Dulbecco's modified Eagle medium.

NA F　S　V　2　neo/Eca　R1 digestate (Southern (S6stearnP ) and Barge, (1982) 7. MoJ.

Appl, Gg%gt, Vol 1. p, 327). mouse carrier -DN A: Isolated from Barb/c macus spleen and pancreas, partially cut Three types of high molecular weight DNA with reduced total molecular weight regarding the effect on cell viability Amplitude settings were tested.

Cells were grown in three separate cultures with these same settings (DMEM5asl psV2 neo/EcoR1 depletion for 10 seconds) sonicated in the presence of 20 μt of mouse carrier DNA and 50 μt of mouse carrier DNA. .

After sonication, the contents of each bijou were transferred to a 6-piece vetri dish and incubated at 37°C for 3 days. , an atmosphere of 100% humidity, 90% air and 0.10% CO under standard tissue culture conditions. Cultured under ambient air.

Cells were then transferred to 9 large containers as described below.

Setting 0 3x9 my petri dish Setting 1’ 3X9cm Petri dish Setting 3 lx9cm petri dish Fresh MEM + 10% calf serum was added to each Petri dish to make a volume of 10- . Antibiotic 0418 was then added to a concentration of 1/wt medium. Next Cells were incubated for 5 days and then transferred to a larger container. this last When I inspected the vetri dishes before transferring, I found that there were a large number of live/replicating birds in the set 30 dishes. Myeloma cells were observed. Fewer viable cells were found in dishes with setting l; No viable cells were found in the Set 00 dish.

Therefore, psV2%-〇 plasmid DNA invades sonicated myeloma cells. did not invade control cells) and was integrated into these cells in a heritable manner. . This plasmid confers resistance to the antibiotic 0418 on sonicated cells. gave. Therefore, the sonicated cells will survive in this antibiotic, whereas the sonicated cells will survive in this antibiotic. Control cells that did not receive lasmid did not survive.

After an additional two weeks the cells were still viable in the antibiotics. This is the above %go Indicates that the gene has been integrated into the cell in a hereditary manner.

Example 2゜ Sonication of DNA to Fibroblast Deafness (So%1fa-Soniprep 150 Ultrasonic diswitch grater MSE CLinbro) 24-well tissue culture plate -70-・Laboratory leeds 9cmM petri dish for tissue culture - NUNC Pbar2 fibroblast-retrovirus packaging system (Reference: SEP:z (C gpko, C, L, et al., Ca1137.1053-1062.1984).

pzzp5v(x), a human N- ZNR3 plasmid consisting of cDNA encoding the rtLj protein (see above) ).

Mouse carrier D A' A: BaLb/c mouse pancreas and liver High molecular weight DNA isolated from Method Psi-2 cells were placed in the wells of the Lympro plate at 9 x 10'/- medium (DME M-Flow Laboratories, 70-Labora containing 10% Danar Calf Sea Serum It was injected in suspension form.

As in Example 1, the tip of the index microprobe of the MSE nib 150 The end was inserted into the center of the well, in this case one depth below the surface of the medium. Each In this case, power was applied to the microprobe for 10 seconds. 3v1 different °amplitude” ( (Read from the scale on the MSE device) Settings for viable cells 0 8.7X10' 1 5.4X10' 3 1.9X10' For the actual experiment, two wells were treated in each setup. Each well has 9X10' cells (initial) plus 10μ2 ZNR3 DNA and 10μ2 It contained mouse carrier DNA. Ultrasonic treatment for 10 seconds at each setting After treatment, 0.2 tnt of cell suspension was removed from each well for counting. cell The numbers were as above (i.e., in this experiment, D NA did not change cell viability).

The remaining cells were allowed to adhere to the bottom of the well, and in this state they were incubated at 37°C with 100% humidity. It was placed in an atmosphere of 9% CO2/91% air.

After 2 days (sonication, day 0 - cell smearing, day 2), trypsin / EDTA solution (Flow Laboratories, 2-/well) was used to add cells to the wells. It was detached from the bottom.

Cells were placed in 2X10 cm tissue culture petri dishes/wells (i.e. in each sonicator setup). 4 pieces per plate).

Add fresh medium 9- to each Petri dish and then place them in a 37°C incubator. and incubated in the same manner as above. However, in this case, the medium is 1!9/ It contained 1R1 G418. Cells that have taken up plasmid ZNP3 have this The plasmid contains the same genes found in the psV2nao plasmid of Example 1. Because it contains the gene, it was resistant to this antibiotic. On day 188, replace the medium. Ta. On day 200, count the colonies of 0418-resistant Psi-2 cells on the Petri dish. (groups of 16 or more cells were considered colonies).

The results were as follows.

This is 10' colony/106 sonicated viable cells/10μ plasmid DNA. 2, that is, 1/10' pieces/μ2.

To achieve maximum efficiency, different cell types require different sonicator settings and It would require five punts.

Procedural amendment (ji force) February 1990 tωd 1.Display of the incident PCT/GB88100738 2. Name of the invention Modification of viable cells 3. Person who makes corrections Relationship to the incident: Patent applicant address Name: AmarJam International-PLC 4, Agent Address: Shin-Otemachi Building, 206-ku, 2-2-1 Otemachi, Chiyoda-ku, Tokyo 5. Date of amendment order: January 30, 1990 (old) 6. Subject of amendment Translated international search report of the specification and claims, typewritten l1llyyB@sal Aa1++n, m,? CTtGB a810073 8SA 24184

Claims (8)

[Claims] 1. Traumatize the cells in the container, in the presence of the substance, or immediately before the addition of the substance. introducing the substance into living mammalian cells by subjecting them to sufficient sonication to , or a method of fusing a substance with the cell. 2. Claims in which the substance introduced into the cell is DNA, RNA or protein The method described in box 1. 3. The substance introduced into a cell is a gene, and the cell then replicates and carries the gene. The method according to claim 2, wherein the method is expressed and transmitted in a heritable manner to daughter cells. . 4. 2. A method according to claim 1, wherein the substance is incorporated into the cell membrane. 5. Ultrasonic excitation treatment of a mixture of two or more homogeneous or different cell types 2. The method of claim 1, which induces cell fusion. 6. Claims 1 to 5, wherein the cells are kept suspended in a liquid. The method described in any of the above. 7. Claims that cells are concentrated in suspension by standing waves induced by ultrasound The method described in item 6 of the scope of 8. During and after the ultrasound excitation treatment, the cells are suitable for reintroduction into the host mammal. The person according to any one of claims 1 to 7, which is held in the form of Law.