AU656520B2 - Process for the artificial stimulation of cells and cyte culture device - Google Patents

Description

il -l' "c i; I- OPI DATE 10/10/91 APPLN. ID) 75770 91 AOJP DATE 07/11/91

PCT

PCT NUMBER PCT/FR91/00200 DEMANDE INTERNATIONALE PUBLIEE EN VERTU DU TRAITE DE COOPERATION EN MATIERE DE BREVETS (PCT) (51) Classification internationale des brevets 5 (11) Numero de publication internationale: WO 91/13977 C12N 13/00 Al (43) Date de publication internationale: 19 septembre 1991 (19.09.91) (21) Numiro de la demande internationale: PCT/FR91/00200 (74)Mandataire: WARCOIN, Jacques: Cabinet Regimbeau, 26, avenue Kleber, F-75116 Paris (FR).

(22) Date de d6p6t international: 12 mars 1991 (12.03.91) (81) Etats designis: AT (brevet europeen), AU, BE (brevet euro- Donnees relatives A la prioriti: peen), CA, CH (brevet europeen), DE (brevet europeen), 90/03108 12 mars 1990 (12.03.90) FR DK (brevet europben), ES (brevet europ6en), FR (brevet 90/03109 12 mars 1990 (12.03.90) FR europ6en), GB (brevet europben), GR (brevet europ~en), IT (brevet europeen), LU (brevet europeen), NL (brevet europeen), SE (brevet europ6en), US.

(71) Deposants (pour tous les Etats d~signs sauf US): INSTITUT NATIONAL DE LA RECHERCHE AGRONOMI- QUE [FR/FR]; 147, rue de l'Universite, F-75341 Paris Publiie Cedex 07 SOCIETE NATIONALE ELF AQUI- Avec rapport de recherche internationale.

TAINE [FR/FR]; Tour Elf, 2, place de la Coupole, La Avant I'expiration du dblai privu pour la modification des Defense 6, F-92400 Courbevoie revendications, sera republihe si de telles modifications sont reues.

(72) Inventeur; et Inventeur/Deposant (US seulement) OZIL, Jean-Pierre [FR/FR]; 4, rue des Fontaines-du-Temple, F-75003 Paris 6 (54) Title: PROCESS FOR THE ARTIFICIAL STIMULATION OF CELLS AND OVOCYTE CULTURE DEVICE (54)Titre: PROCEDE DE STIMULATION ARTIFICIELLE DE CELLULES ET DISPOSITIF DE CULTURE D'OVO-

CYTES

(57) Abstract The present invention concerns a process for artificially stimulating a group of cells reproducing the effect of the biochemical regulation mechanism, controlled in natural physiological conditions by an internal oscillator, comprising the steps below: a) the cells are placed in in vitro culture for a given time; b) the ells are placed in the pulsing medium after the culture medium has been removed; c) the cells are subjected to pulses generated by an electric field; d) the cells are once more placed in the culture medium; e) the previous stages are repeated a certain number of times; f) cells in a uniform active state are obtained. It also concerns a device for the culture of ovocytes facilitating the operation of the process.

(57) Abrgig La pr6sente invention concerne un proc6de de stimulation artificielle d'un ensemble de cellules reproduisant I'effet d'un mecanisme de regulation biochimique, contr616l dans les conditions physiologiques naturelles par un oscillateur interne, caractdrise en ce qu'il comprend les 6tapes suivantes: a) les cellules sont plac6es en culture in vitro pendant un temps determin&, b) les cellules sont plac6es dams le milieu d'impulsion, apr&s elimination du milieu de culture, c) on soumet les cellules A une impulsion genbre par un champ 6lectrique, d) les cellules sont A nouveau places dans le milieu de culture, e) on rpete les tapes pric&dentes un certain nombre de fois, f) on obtient des cellules dans un 6tat active homog6ne. Elle concerne 6galement un dispositif de culture d'ovocytes permettant la mise en ceuvre du proc6d6.

"c 1 The present invention relates to a method for the artificial stimulation of cells, especially oocytes, the said stimulation being capable of modulation with the passage of time, and to a device for the culture of cells enabling the method to be carried out.

The present invention relates to the stimulation of cells, especially oocytes or fertilised eggs, by a biochemical mechanism regulated under physiological conditions by an internal oscillator.

The method in question is, in particular, a procedure for the cloning of animal embryos, and especially for obtaining recipient oocytes competent for the transplantation of a cell nucleus in a homogeneous state.

The cloning of embryos of domestic animals is the method enabling the genetic variability induced by fertilisation to be remedied, and the genetic improvement of a breed to be standardised.

During fertilisation, the entry of the spermatozoon will have two major functions: provision of the male haploid genome, and activation of the development which restructures the male nucleus by the cytoplasm of the oocyte and promotes nucleus-cytoplasm interactions.

On average, 12 to 20 hours elapse between fertilisation and the first cell division, during which time a combination of phonomena takes place, both paternal and maternal genomes having complementary roles for subsequent development of the embryo (Surani et al. 1984, I' Nature 308, 548-550), but the exact mechanism of this is not known.

The cloning of an embryo is a method directed towards obtaining the largest number of living animals following the transfer of cell nuclei from this embryo (which contains several cells) into enucleated and activated oocytes. The two functions of fertilisation are i split. In order to obtain subsequent development of the egg, it is necessary to undertake an activation of the recipient oocyte before nuclear transplantation. The activation can also be applied to the initial hours of S k- 0-1 -2the development of the fertilised egg.

It is known that the eggs of mammals may be activated artificially by various physical or chemical stimuli, including electric, thermal or osmotic shocks, enzymes or anaesthetic agents (Kaufman MH 1983 Early mammalian development parthenogenetic studies Cambridge University Press). However, the activation is always identified with a single stimulus, limited in time, assumed to mimic the penetration of the spermatozoon into the egg. None of these treatments reproduces the series of physiological changes taking place in the egg after the penetration of the spermatozoon.

Only a few cases of parthenogenetic activation of the cow oocyte are known (Menezo et al. 1976 Commission of the European Communities, Agricultural research seminar, Egg transfer in Cattle. Eur 5491); no truly reliable and precise method of activation of bovine oocytes is available. Experimental activation with ethanol has many drawbacks: wide variability of the results depending on the age of the oocytes (Cuthbertson, 1983; J. Exp. Zool. 226, 311-314).

As regards the cloning of embryos, the first undisputed results have been obtained by S. Willadsen in 1986 in ewes. They were followed by those of Prather et al. in 1987 in cows and in 1989 in sows. In rabbits, the first individuals derived from cloning were obtained by Stice and Robl in 1988. The success rates of these experiments, reported in the publications, do not generally exceed However, American companies (Granada Genetics, Houston, Texas) or Canadian companies (Alta Genetics, Calgary, Alberta) are appearing to master the cloning procedure in cattle. It is stated that around one hundred calves are already considered to have been obtained on the North American continent. This industrial I take-up reflects the interest aroused by embryo cloning in cattle. However, have the techniques really progressed? The procedures described in the publications V\A i resemble one another. The oocytes used have undergone a is I T '41 3 period of ageing of 6 to 10 hours. This ageing is made necessary in order to promote activation (no conventional activation technique enables freshly ovulated oocytes to be activated, even in cattle (Ware et al. 1989)). The chromosomes organised in metaphase II are removed "blind" in the region of the first polar body, but techniques of visualisation of the chromosomes by fluorescence are employed. An embryonic cell originating from a morula is introduced under the zona pellucida and fusion is obtained by pulses from electric fields. Activation of the oocyte is generally induced by the cell fusion procedure; in some cases, it is not described. Two investigators were able to obtain a lamb following the transfer to an oocyte of a nucleus originating from an embryonic bud cell (Smith Wilmut, 1989).

However, the level of the results and the complexity of the mechanisms involved, such as the stage of differentiation of the nucleus, the phase of the cell cycle, the state of the recipient oocyte, its age and the activation procedure, do not permit the reason why some embryos develop and others do not to be understood. Many theories to explain the failures, such as that based on the transcriptional activity of the transplanted nucleus, have been contradicted by some experimental results.

New data have appeared regarding the physiological mechanisms triggered by fertilisation, and in particular as regards the rhythms of the rariations in the level of free calcium and of the second messengers such as inositol 1,6,5-triphosphate (InsP3) (Cuthbertson et al. 1981; Cuthbertson Cobbold, 1985; Miyazaki et al.

1986; Miyazaki, 1988). The periodic activity of these second messengers could regulate the syntheses of nucleic acids (DNA and RNAs) and proteins (Basset et al. 1968; Rodan et al. 1978).

The process is triggered by the spermatozoon in the seconds following fertilisation. The chain of reactions leading to the production of InsP3 appears to depend on an influx of calcium ions. The InsP3 binds to a specific recipient which controls the activity of a 1

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calcium channel located on the intracellular membrane of an intracellular calcium reservoir. The calcium thus released activates specific proteins, which themselves activate specific processes, for example calciumcalmodulin complexing, kinase activation, and the like.

Calcium is considered to be the main activator of the metabolism. This cycle of reactions is reproduced at a frequency of the order of one minute for several hours (McCulloh et al. 1983; Igusa Miyazaki, 1983-1986; Miyazaki et al. 1986; Miyazaki, 1988).

This rhythmic activity, due to a system of intracellu3ar signals emitted at an appropriate frequency, appeas to depend on the presence of male and female pronuclei, since it has not been possible to observe it on parthenogenetic eggs activated by conventional methods (Cuthbertson et al. 1981; Cuthbertson Cobbold, 1985; Miyazaki, 1988).

The injection into oocytes of a G protein (guanosine 5'-0-(3-thiotriphosphate) (GTP[S]) induces several cycles of calcium release, but does not enable this activity to be maintained beyond the fourth (Swann, Igusa Miyazaki, 1989).

The respective genetic contributions of the oocyte and the spermatozoon are very well documented in the mouse. These studies show that the normal development at term of an embryo depends on the permanent presence throughout the first cell cycle of the two parental pronuclei (PN) (Surani Barton 1983; McGrath Solter, 1984; Surani et al. 1986; Mann Lovell-Badge, 1986, 1988)..

S3/The production of young by cloning can be carried out in practice only if reliable and reproducible methods are available. The quality of activation of the egg plays an important part, hitherto ignored, in the development.

Control of the activation, understood as a rhythmic phenomenon extending throughout the first cell cycle, is essential, not only for the routine production of activated and synchronous oocytes, but also so as to enable "cloned" eggs to benefit from this type of imposed ,2 5 1 p. activation in order to develop normally.

The present invention hence relates to a method for the artificial stimulation, in particular in the long S |term, of an assembly of cells reproducing the effect of a biochemical regulation mechanism controlled under natural physiological conditions by an internal oscillator, characterised in that it comprises the following steps: a) the cells are cultured in vitro for a specified time, b) the cells are placed in the pulsating medium after removal of the culture medium, c) the cells are subjected to a pulse generated by an' electric field, d) the cells are placed again in the culture medium, e) the above steps are repeated a number of times, and f) cells are obtained in a homogeneous activated state.

Removal of the culture medium will preferably be supplemented by a washing of the cells with the pulsating medium.

20 This stimulation method, although it may be used in a large number of methods, has been developed in the context of the activation of oocytes for the purpose of cloning embryos. Recent studies have, in effect, shown that in mammalian eggs, fertilisation is accompanied by a transient increase in the intracellular concentration of free calcium ([Ca 2 followed by a series of fluctuations of this concentration lasting at least 4 hours.

It is not yet known by what means the spermatozoon triggers these variations in as well as the A 30 exact biological functions of these long-lasting fluctuations of Ca 2 However, they appear to be characteristic of fertilised eggs, and have never been observed when oocytes are activated artificially (Miyazaki 1988 J. Cell. Biol. 106, 345-353).

The method developed in the present invention will hence enable oocytes to be stimulated periodically by influxes of calcium at plasma -nembrane-lg-1vel.

In effect, .by, introducing-- a----ions into the Spulsating medium, these ions will be able to enter the L 1 i,_a L~ 1 -i -6 cell by electroporation, that is to say the creation of "pores" generated by the electric pulse.

Beyond the immediate phenomena induced in the oocyte, such as expulsion of the polar body, the effects of this stimulation will manifest themselves on the subsequent stages of the development of the embryo, even though the treatment has stopped. Thus, the phenomena of compaction and cavitation may be affected.

This process may be applied to freshly ovulated oocytes, obtained by hormonal stimulation of the female.

The activation of oocytes under defined conditions of frequency, intensity and duration causes expulsion of the second polar body. It is then possible to withdraw the chromosomes which are to be found at the end of telophase immediately under the second polar body. Oocytes are obtained in the same physiological state possessing a pronucleus.

A blastomere is then introduced under the zona pellucida, and cell fusion is performed by the action of an electric field.

The method may then be applied to the cloned embryos thereby obtained, in order to improve their development, that is to say to regularise the cell divisions and obtain compaction.

Stimulation by this method may be applied to freshly ovulated oocytes placed in the presence of an inhibitor of the expulsion of the second polar body, such as cytochalasin B, which maintains a diploid state. A i population of parthenogenetic embryos is thereby obtained, which may be implanted into recipient females and 4- will exhibit a synchronous development of the foetus and the embryonic appendages.

These experiments, carried out first on rabbit oocytes, have been confirmed on mouse oocytes, renowned for being refractory to activation immediately after ovulation. The efficacy of the method is hence reproducible from one species to another. The method of activation may definitely be applied to freshly ovulated cattle or sheep oocytes.

;1 P- L 7 If the oocytes are withdrawn during treatment, they regress and some artefacts are obtained, such as oocytes in metaphase III reproducible at will, and which offer novel possibilities of study, in particular on the dynamics of the cytoskeleton and the activation of the genome.

The method has been devised on the basis of the effects ofelectric fields on plasma membranes.

It has been shown (Zimmermann, 1982) that pulses from electric fields of the order of 1 to 3 kV.cm and lasting a few ps can create micropores in the plasma membrane and ca:i establish a direct communication between the intra- and extracellular media. It has thus been possible, for example, to cause calcium to enter sea urchin oocytes by exposing them to pulses from electric fields in the presence of calcium ions (Rossignol et al.

1983). The amplitude of these influxes may be modulated by the duration of the electric-field pulse.

The method involves the combination of a method for the in vitro culture of a batch of oocytes (or embryos) by perfusion between two electrodes, and a method of washing with a non-conducting solution containing 0 to 30 pM calcium, in which the pulses are delivered. The presence of a very weakly conducting solution, for example an isotonic glucose solution containing Ca2+ ions at the time of a pulse enables an electric field to appear between the electrodes. An excessive presence of ions decreases the "electric field" 1 effect.

30 Preferably, the concentration of Ca 2 ions is between 8 and 20 pM. Good results are obtained, for example, with a concentration of Ca 2 ions equal to 10 pY or alternatively 16 pM. The alternation of a period of culture and a period of washing enables the oocytes to be subjected frequently to stimulations in a very well defined ionic medium.

This method may be extended to the stimulation of 'IU cells by a whole range of simple or complex ionic or 0 molecular signals at.variable concentrations which will i <h bA 8 be determined by the composition of the pulsating medium.

This method enables the frequency of the signal to be modulated by the time elapsing between two washes, and its amplitude by the duration of the electrical pulse.

It is imperative to replace the culture medium complete.'y by the pulsating solution, since the intensity of the current due to excesses of ions originating from the culture medium would destroy the oocytes. Immediately after pulsing, the pulsating solution is replaced by the culture medium, since the oocytes are unable to survive in the pulsating solution.

This method may be controlled via software which will enable stimulation treatments to be created according to speciL equations, exponential, sinusoidal, Fourier series or the like.

One of the aspects of the present invention is the simultaneous treitment of an assembly of oocytes, which will thus be obtained activated in the same physiological state. Nuclear transplantations may thus be carried out on physiologically identical oocytes.

During the cloning, the sequence and the phases of the cell cycle during which the operations are interlinked have very important consequences on the remodelling of the nuclei and on subsequent development.

It 'is extremely advantageous to be able to standardise the various phases of the treatment of the oocytes in order to obtain a good reproducibility of the method.

The present invention hence relates to a dynamic system operating in continuous fashion and capable of being automated, carried out by a device permitting the automatic succession of the washing and stimulation steps and the acquisition of the stimulation parameters.

This device is characterised in that it provides for immobilisation of the cells during the various phases of the treatment.

Different embodiments of this device are seen in Figures 1 and 3.

4 kiK 9I It consists of a chamber containing at least one fluid inlet (10, 11) and one fluid outlet and is characterised in that, at the bottom of the chamber, there are one or more orifices whose geometry is such that it opposes the passage of the cells, and in that the liquid entering the chamber is withdrawn, at least partially, through the orifices, creating a partial vacuum such that it substantially provides for clumping of the cells on the orifice or orifices, a portion of the fluid being discharged by overflow.

This device enables the cells to be retained without damaging them or introducing a spurious mechanical stress. In addition, it enables cells to be put back, withdrawn or moved at any time. This device hence enables cells to be placed successively in media of chemical composition of different natures, during variable periods and according to a variable frequency.

The chamber contains two electrodes, preferably parallel, on its internal walls, and the orifice or 20 orifices are aligned at an equal distance from the electrodes, immersed in the medium corresponding to the phase of the treatment in progress.

By means of this device, it is possible, at will, to culture the cells or alternatively, in the pulsating medium, to cause an ion, a molecule or a complex biological or chemical substance to enter the treated cell.

This device makes possible, in particular, the treatment of an assembly of oocytes, in large number, in the same chamber without it being necessary to place them in separate compartments.

In particular, the device contains at least two fluid inlets, which may be superposed.

The discharge of the fluids may also be carried out l' erally. I A different injection tube is associated with each medium admitted successively to the chamber.

At the time of injection of the pulsating medium, a suction system fitted to the culture medium inlet device permits suction of a small amount of the pulsating 1 r i r ii r 11 ir~ i 1I I 10 medium, which enters the culture medium inlet pipe; entry of ions of the culture medium into the chamber during washing is thereby avoided. The passage of the flows of medium is shown by arrows in Figure 3.

These media, both the culture medium and the pulsating medium, flow continuously, and this flow of liquid by a suction effect provides for maintenance of the oocytes between the electrodes during renewal of the medium.

The compositions of the culture media are known and depend on the cells being cultured. The pulsating medium generally consists of a non-ionic medium in order to provide for a field effect. It will be an isotonic solution, for example, of glucose.

The presence of a large amount of ions can seriously harm the cells during pulsing. It is hence necessary to wash the cells with the pulsating medium in order to remove the ions contained in the culture medium.

This device may be designed as an activating chamber whose capacity may be adapted to the number of cells which it is desired to treat simultaneously.

The characteristics of the method employing the device can vary widely, and are, in general, limited only by conditions such as the washing times. Thus, the pulse frequency is limited by the minimum washing time with the pulsating medium.

Parameters of tbi method and of the device, that i is to say the fluid inlets and outlets, like the frequency, duration and intensity of the electrical pulses reaching the electrodes, are controlled by an electronic system.

The characteristics of the electrical pulses ~themselves can vary and can depend on the cells and on I the objectives desired.

In general, the electric fields vary from 1 to 3 kV.cm' 1 and the electrical pulse lasts from 10 As to 2,000 As.

AC vary during the different steps of the same process of vay Tefrqec addrtono h ple a i 1- 11 stimulation.

The organisation of the tubes which open into the chamber enables media to be injected without the oocytes being detached by the gas bubbles which ultimately form in the pipes. In addition, the distribution structure of the media enables ionic contamination of the stimulation medium with ions of the culture medium to be limited considerably.

The pumps for injection of the media may be controlled by a microcomputer, which will determine the repeatable washing sequences while maintaining the cells between the electrodes.

This device may be applied to the treatment of cells with any substance which it is desired to cause to enter the intracellular space. The composition of the pulsating medium will determine .the entry of this substance according to a concentration gradient.

During cell culturing, it will be possible to induce entry of the desired substance into the assembly of cells at a specified time, which can be unique. For this purpose, the culture medium will be replaced by the pulsating medium and the electric-field pulse will be set off, the duration of the pulse determining the amount of substance which enters. This pulsating medium may then be discharged and the cells set up again in culture in a suitable medium. The advantage of this system is that it permits the simultaneous treatment of an assembly of cells, which are thereby synchronised. It operates continuously and avoids all cell manipulation, thereby providing for better reproducibility and greater speed of execution. This permits a standardisation of conditions and facilitates the transfer of technologies.

It is possible to envisage causing an ion or simple molecule to enter cells. This device can also be applied to the stimulation of cells with a more complex molecule, or to the entry of episome or DNA fragments.

This device will be used to carry out a cyclic stimulation of cells, several basic sequences being ,?CL carried out successively, the intensity, duration and so 12 frequency of the pulses being controlled via a microcomputer.

This device enables the method of activation and F synchronisation of oocytes to be carried out by repeated 5 stimulations with Ca 2 for the purpose of cloning embryos of domestic animals.

An embodiment of the chamber is shown in Figure 1.

The oocytes 1 are placed on a rectilinear slit 4, produced by the juxtaposition of two glass plates 3 and the separation of which is less than the diameter of the oocytes. The media, culture medium and pulsating medium, are admitted at the top of the chamber, each via a different tube, 10 and 11, respectively, avoiding reciprocal contamination.

The medium 9, corresponding to the treatment phase in progress, is admitted in continous fashion and is discharged via a tube 6 situated at a lower level than that of the oocytes. This stream of liquids maintains by suction the oocytes flattened on the bottom of the chamber.

On the internal walls of the chamber, there are two parallel platinum electrodes 2 and 7, 1 cm long.

The chamber 8 is thermostated at 38°C.

Each medium is admitted to the chamber after passage via a system retaining large gas bubbles.

This system consists of a glass flask to which the liquid is admitted. A gold shaft perforated with holes of small diameter leaves this flask; after passage through this shaft, large gas bubbles are retained and the liquid is admitted to the chamber via a short tube.

Female rabbits were superovulated by injection of FSH and LH and the oocytes removed from the oviducts.

After treatment for 5 min with hyaluronidase, the oocytes are placed in the activating chamber in B2 medium (Menezo, 1976 C. Hebd. Seanc. Acad. Sci. Paris 282, 1967-1970) at 38"C, in an atmosphere containing 5% of CO 2 The oocytes may be subjected immediately to the activation treatment; no period of ageing is necessary.

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25 4. ;r i: 13 They are subjected for 90 minutes to a series of electric-field pulses, at a rhythm of one pulse every 4 minutes, equivalent to 22 pulses of decreasing duration dissipating a total energy of 1250 pJ, lasting a total of 14,868 ps, the electric field having a value of 1.8 kV.cm 1 Before each pulse, the culture medium is replaced by a pulsating medium consisting of isotonic glucose solution containing 10 uM CaCl 2 At the end of the activation treatment, all the oocytes have two well-formed polar bodies. A homogeneous population of activated oocytes in the same physiological state is hence obtained at the same moment, to within a few minutes. This has two advantages: the first is to be able always to carry out nuclear transplantations in physiologically identical eggs, and the second, more useful, is to be able to work at the time when the chromosomes (haploid) are all at the end of telophase immediately under the second polar body. This natural localisation of the place where the chromosomes occur facilitates their withdrawal "blind", and enables the manipulation steps to be interlinked rapidly without resorting to fluorescent labelling techniques.

Withdrawal of the chromosomes may thus be performed. It will be noted that the two polar bodies are not always beside one another (some are opposite to one another; for this reason the first polar body is not a good marker of the place where the maternal chromosomes occur). The efficiency of this operation, checked by the subsequent presence of a pronucleus, is greater than A blastomere is then introduced under the zona pellucida.

to 20 oocytes may be handled in 1 hour.

Cell fusion is then performed. The cell fusion method is derived from the work of Zimmermann on electric fields. The procedure has been refined (Ozil and Modlinski 1986, J. Embryol. Exp. Morph. 96, 211-228).

100% fusion is obtained. It will be possible to carry out the alignment and cell fusion in the stimulation chamber automatically, under the control of 1

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\a 14 14 software. This procedure will enable the duration of the manipulations to be limited and the experimental conditions to be standardised.

EXAMPLE 1 MATERIALS AND METHODS Superovulation was induced in sexually mature female rabbits of a variety of species by subcutaneous injection of follicle stimulating hormone (FSH) and luteinising hormone (LH) according to the technique described by Kennely and Foote (1965) and modified by Thibault (personal communication). The females received 2 mg of FSH in five injections at 12 hour intervals: 0.250, 0.250, 0.650, 0.650 and 0.250 mg. 12 hours later, prior to mating with a vasectomised male, they were injected with 0.33 mg of LH.

Oocytes were recovered from the oviducts 12-15 h after mating by washing with phosphate buffered saline (PBS) solution. They were incubated for 5 minutes in hyaluronidase.(300 i.u.ml'- in PBS) to remove the fcllicular cells. After the treatment, the oocytes were cultured at 38'C in B2 medium (Menezo 1976) in an atmosphere containing 5% of COz.

Method and experimental procedure The membrane permeability of freshly ovulated rabbit oocytee was transiently increased by opening pores with a pulse induced by an electric field in the presence of 10 pM Ca 2 contained in a 0.3 M glucose/18 MOhm HzO solution (pulsating medium). It is accepted that, while the pores are open, a flux of ions passes according to the concentration gradients through the cell membrane into the cytosol, as has already been shown in sea urchin oocytes (Rossignol et al. 1983). Thus, the ionic influx may be adjusted under these conditions, either by the difference in ionic concentration between the inside and the outside of the cell, or by the duration of the pulses.

The experimental procedure for an electrically induced ionic flux was similar to that described previously for the fusion by an electric field of two-call cA i AL1 activation can also be applied to the initial hours of 11 1 rabbit embryos (Ozil and Modlinski, 1986). The oocytes are cultured with M16 medium (Whittingham, 1971) at 38 0

C

in a specially designed chamber. Before each pulse, the culture medium is automatically replaced by the pulsating medium. The details of the chamber are described in Figure 1. Each pulse was composed of two alternating pulses in order to avoid "lateral electrophoresis" (Jaffe 1977) of membrane proteins, which might occur after several pulses of the same polarity (Poo, 1981). The amplitude of the ionic signal was modulated through the duration of the pulse. The whole process was controlled by an IBM PC-AT 286 microcomputer via a Tektronix MI 5010 interface with a program written in MS-BASIC. The actual voltage and the current between the electrodes were measured with a Tektronix 77041 oscilloscope mounted with a 7D20 programmable digitiser and a 7A22 amplifier.

The rhythm of electrical pulses and the total duration of the treatment were the same for all treatments, that is to say, applying a pulse every 4 minutes for 90 minutes. These values were chosen because they fit well with the frequency and average duration of hyperpolarisation of the membrane potential of rabbit oocytes during fertilisation (22 biphasic fluctuations of the membrane potential during the first 90 minutes following sperm-egg fusion, one pulse every 4 minutes McCulloh et al., 1983). The variation of the membrane potential is known to reflect the passage of K based on calciumactivated channels, and hence to constitute a reliable indicator of [Ca 2 (Miyazaki and Igusa 1982). The amplitude of electric fields (1.8 kV.cm 1 was constant I for all experimental groups.

Oocyte treatment The effects of the different treatment parameters on oocyte activation were studied in four experimental groups.

Group A Experimental environment In order to test the effect of the experimental conditions (continuous perfusion, replacement of the .1 C- j A 1 resemble one another. The oocytes used have undergone a .iN I 16 culture medium and electrical pulses), freshly ovulated oocytes and fertilised eggs were subjected to 22 double pulses of 900 is in the pulsating medium devoid of Ca 2 (the first pulse is given 13 to 15 hours after mating).

After the treatment, the fertilised eggs were transferred to female recipients to determine survival to term.

Unfertilised eggs were cultured in vitro and the rate of parthenogenetic activation was noted.

Group B Calcium ions and pulse duration The effect of pulse duration was studied in the pulsating medium containing 10 1m CaClz. The treatment for which oocytes are not activated was considered to be the minimum duration, and that resulting in oocyte lysis was considered to be the maximum duration. A set of six treatments with 22 constant double pulses was chosen arbitrarily. These treatments have a pulse duration equal to 200, 300, 600, 900, 1,200 and 1,500 ps, respectively.

The effect of the presence of Na and Mg2+ ions at a concentration of 10 1M in the pulsating medium was tested with 22 constant double pulses lasting 900 ps.

Group C Pulse modulation and type of parthenogenetic activation The treatment with 22 double pulses of constant duration reveals the value of the pulse duration for which the maximum and minimum effects are recorded. These constant treatments do not produce a high level of activation with a uniform type of parthenogenetic egg. In order to check whether a gradual reduction of the calcium stimuli in a given treatment has an effect on the type of parthenogenetic reaction, oocytes were subjected to treatments in which the pulse duration decreased gradu- 1 ally according to a negative exponential relationship.

Four treatments were tested in accordance with the maximum duration of the first pulse. Figure 2 shows the graph of the pulse durations for these treatments.

A negative exponential relationship (D(u)-e (axutb)+c) between the pulse index (1 to 22) and the A i

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-17 pulse duration (1,500 As to 200 AS) was chosen arbitrarily in order to f ind the value of each pulse during the treatment.

With D(u) pulse duration of the cycle The coefficient a determines the slope of decrease of this relationship.

The coefficient b determines the duration of the first pulse.

The coefficient c determines the duration of the final pulse.

The coefficients a and c are constant and equal, respectively, to -0.4 and 200.

b 5.9920 for treatment I 6.5510 for treatment II 6.9080 for treatment III 7.1700 for treatment IV.

Group D Modulation of the electric field and development in vitro Eggs are treated in the presence of 8 pg m1- 1 of cytochalasin B in the culture medium to block expulsion of the second polar body and obtain a uniform population of diploid parthenogenetic eg~gs. Two treatments were applied to group C, treatment I which is the weak treatment with a total pulse duration equal to 11,228 As and treatment III which is the strong treatment lasting 14,868 A. Parthenogenetic eggs were cultured in. vitro to the blastocyst stage and the influence of the two treatments was evaluated by the rate of blastocyst formation.

30 Post-implantation viability of diploid parthenogenetic embryos Embryos at the 4-cell stage weze transferred to the oviducts of female recipients. Fertilised eggs were also transferred to the opposite corn of some recipients in order to compare parthenogenetic development to n'ormal development. The female recipients were autopsied between day 8.5 and day 13 of pregnancy and the number of sites of implantation of live foetuses was noted.

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4 enable "cloned" eggs to neneriT rroum uLi. -l 18

RESULTS

Group A Effect of the experimental environment (controls) When the pulsating medium contains no electrolytes, none of the oocytes (105) is activated. The experimental environment and the culture conditions, that is to say replacement of the culture medium before each pulse by a pulsating medium not containing electrolytes and treatment with relatively strong alectrical pulses (2 x 1.8 kV.cm 1 x 900 As x 22 times, that is to say a total pulse duration of 30,600 As), has no visible effect on freshly ovulated oocytes, but the conditions do not permit development to be triggered. In contrast, 41% (9/22) of fertilised eggs treated in the same manner developed to term, thereby demonstrating that treatment with high voltage electrical pulses has no contrary effect on the capacity of fertilised oocytes for development. It was not possible to replace the culture medium completely by the pulsating medium before each pulse. The current measured during pulsing reveals that the conductivity of the pulsating medium is at least 15% higher than that of the pulsating medium measured between the electrodes before the experiment and before the first injection of culture medium. During the experiment, ions originating from the culture medium are still present during pulsing, and this modifies the ionic signal. These variations in the micro-environment do not appear to have any significant effect on activation or embryonic development. In this series of experiments, the eggs are washed with the pulsating medium for 45 seconds every four minutes. This period during which the eggs are not in the culture medium has no significant effect on the activation or on survival to term.

Group B Calcium ions and pulse duration Table I summarises the results of the experiments in which the rate of activation was tested in relation to o n f the pulse duration in the presence of 10 AM CaC1 2 duigplig n ti oiisteini inl hs .1 vriaion in he icr-enironentdo ot apea tohav an sinifianteffct o acivaton r ebryoic eve 19 19 The appearance of pronuclei after 3 or 4 hours of culture acts as a marker for parthenogenetic activation.

These results show clearly that parthenogenetic activation is triggered when the pulsating medium contains 10 pM Ca In addition, the rate of activation is directly linked to the pulse duration, which indirectly controls stimulation by calcium. The duration of the ionic stimuli does not influence only the rate of activation, but also the nuclear configuration of the parthenogenetically activated oocytes of similar postovulatory age.

The longer the pulse duration, the higher the proportion of activated eggs, but the greater the proportion of oocytes containing micronuclei.

Group C Modulation of pulses and type of parthenogenetic activation The results are summarised in Table II. The relationship between the type of parthenogenetic activation and each treatment is also shown in Table III. When the pulse duration is reduced, all the oocytes are activated, and the majority of them have a single pronucleus and two polar bodies when meiosis has ended.

Thus, modulation of the calcium stimuli through pulse duration appears to be effective and influences the first stages of development.

In this study, the age of the oocytes was similar, and hence cannot affect the results.

Group D Effect of modulation of the electric field on the in vitro development of oocytes activated by parthenogenesis The results are summarised in Table III. No visible difference in the development of each group could be noted up to the third division.

In parthenogenetic embryos produced by treatment I, a smaller proportion of embryos exhibits compaction, and those which compact are irregular. In contrast, the S\ majority of embryos produced by treatment III compact and L 1 iJs 20 develop to the blastocyst stage.

These results show that the form of the activating stimulus has a profound effect on the capacity of parthenogenetic embryos to develop to the blastocyst stage in vitro.

Post-implantation viability of diploid parthenogenetic eggs 13 female recipients became pregnant and were autopsied between day 8.5 and day 13. The results are summarised in Table IV. Although the parthenogenetic embryos are smaller than the control embryos, they appear morphologically normal according to the criteria defined for rabbits. The ratio between the size of the embryonic appendages and that of the foetus is roughly equivalent for the embryos obtained by fertilisation or by parthenogenesis.

Thus, it appears that the general development of parthenogenetic embryos, the foetus and its trophoblast tissues is delayed.

The dead foetuses have several abnormalities and it was not possible to classify them.

Table I Effect of the presence of calcium ons and electrical pulse duration on the activation of rabbit oocytes Pulse Number Number of oocytes activated 4 duration of lysed acti- with oocytes vated 1PN+PB1 2PN 2PN# PB2 +PB1 +PB1 200 ps 47 0 13 83 17 0 300 ps 99 0 62 68 25 7 600 ps 85 0 98 71 19 900 ps 63 0 100 62 14 24 1,200 ps 97 5 100 57 29 14 1,500 ps 50 3 100 30 17 53

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ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. FR 9100200 SA 46023 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report.

The members are as contained in the European Patent Office EDP file on 16/07/91 I l- n s nt ,hi nrlurnc of information.

21 Table II Effect of modulation of the electric field on activation ~Teatment Numiber of Qocvtes activated with activation lpN+pB1 2PN 2PN# PB2 +PB1 +PB1 1 86 88 83 9 1I 151 99 89 9 111 118 100 91 9 TV 96 100 70 .Abrnrmal prioimclei., sma~ll nicroriclei Table III Effect of modulation of the electric field on lopment of parthenogenetic eggs in vitro Treatment Number of Number Number ooye activa- cultured morulae 8 1 the deve- Number blastotion ted 25(36) 244(100) 1 98 91 III352 100 Table IV Post implantation development subjected to treatment III.

Day ofut~ D 8-9 D 9-10 Number of 2Mc~ transfere 21 26 Number of jinlantations 9 3 cumulative (42.8) (21.2) Nubrof live foetuses 8 3 cumulative (88.8) (91.6) EXAMPLE III PARTHENOGENETIC DEVELOPMENT 23 (33) 216 (88) of parthenogenetic eggs D 10-11 D 12-13 Total 50 15 (27.8) 7 (66.6) 68 23 (30.3) 0 (36) Oocytes freshly collected (13 hi after mating with a vasectomised male) are subjected to a standard comprising 22 pulses (one every 4 minutes) total duration is equal to 22.475 ps (working lex 1370).

The calcium. concentration in the trea.tment and whose reference: pulsating

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V RAPPORT DE RECHERCHE INTERNATIONALE Osmnarile international.p N, PCT/FR 91/00200 ICLASSEMENT DE LINVENTION (si piusieurs symboics dle classification sont aoplicables. l[as indiquer tous)7 Solon Is classification Internationals des brevets (CIB) ou A In fois sian Ia clasaification nationals at Is CIS I M r 5 C 12 N 13/00

I:

22 solution is the only variable. Five values were chosen: 12, 14, 16, 18 and 20 qM of calcium. The table below sum'marises the results.

NIs-

I

(2$ ,Calcium No of Nc:. Nti. eggs No). Preg- No. of N. of hyp1an- lbo. Foetus Live transfers eggs per reci- reci- naricy eggs on inp1an- tation f oetuses rate foetus pient pients rate pregnant tations rate rate pregna-nt recipients FM 2 61 31 1 50% 31 2 6% 1 3% 3% A 3 79 26 3 100% 79 29 37% 16 20% 0% 14 IM 4 90 23 4 100% 90 28 31% 19 21% 7% 16 IM 7 89 13 6 86% 81 31 38% 21 26% 23% 18 M 4 85 21 1 25% 30 3 10% 3 10% pM 3 60 20 2 67% 49 9 18% 5 10% 0% !Ibtal 23 464 20 17 74% 360 102 28% 65 64% 28%

~LL

b-i" I 23 Of a total of 23 transfers, the overall pregnancy rate with all treatments merged is 74%. Among recipients which became pregnant, the implantation rate recorded on day 1.5 of pregnancy (the duration of pregnancy in rabbits is 29 days) is 28%.

The presence of a foetus could be observed in 64% of cases and 28% of them were still alive at the time of autopsy. These results are markedly superior to the results obtained by conventional methods: in mice, the production of a foetus at d 11 is very rare.

However, the most important result lies in the effect of the calcium concentration at the time of pulsing on post-implantation development. Figure 2 shows clearly that the implantation and survival rates at d 11 depends directly on this parameter. Optimum development was obtained with a concentration of 16 pM. In this case, we obtained a record implantation rate of 38% (31 implantations out of 81 eggs transferred), including 26% of foetuses. Morphological comparison of the various foetuses obtained reveals that the foetus are largest in size with 16 pM calcium.

Moreover, we observed that the presence of residual culture medium in the proportion of 1/10,000 in addition to the 16 pM calcium is capable of considerably modifying development, since the embryonic appendages develop but the foetuses are virtually non-existent.

The washing parameters must hence enable the presence of residual culture medium to be limited at the time of pulsing. The presence of residual medium increases the number of ionic species capable of entering the oocyte at the time the membrane is electrically rendered permeable.

EXAMPLE III ACTIVATION OF THE FRESHLY OVULATED MOUSE OOCYTE Mouse oocytes freshly ovulated (12 h post-HCG) are subjected to treatments comprising 33 pulses, one every 4 minutes during 2 hours 12 minutes.

The treatments are carried out with calcium concentrations in the pulsating solution equal i.

I.

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Ss i"frr'~ -i 24 successively to 4 pM, 8 uM, 12 pM and 16 pM, and with variable pulse durations.

The dynamics of formation of the pronucleus may be controlled as a function of the calcium concentration in the pulsating solution and the total duration of electrical pulses (Vitullo and Ozil, 1991).

Claims (23)

1. 2. Method according to Claim 1, characterised in that it is applied to freshly ovulated animal oocytes.
2. 3. Method according to one of Claims 1 and 2, characterised in that the pulse originates from a field of 1 to 3 kV.cm' 1
3. 4. Method according to one of Claims 1 and 2, characterised in that the electric-field pulse lasts from As to 2,000 /s. Method according to one of Claims 1 to 4, charac- terised in that the pulse frequency is modulated by the time elapsing between two washes.
4. 6. Method according to one of Claims 1 to 5, charac- terised in that the frequency and duration of the pulses can vary during the different steps of the same stimula- tion process.
5. 7. Method according to one of Claims 1 to 6, charac- *terised in that the pulsating medium consists of a substantially non-ionic medium.
6. 8. Method according to one of Claims 1 to 7, charac- terised in that the pulsating medium contains an ion or a mixture of ions, a molecule or a complex biological or l chemical substance, which is to enter the treated cell.
7. 9. Method according to Claim 8, characterised in that the compound entering the cell has a messenger role. Method according to one of Claims 1 to 9, -I 111-~ PI 26 characterised in that the pulsating medium consists of an isotonic glucose solution and contains Ca 2 ions.
8. 11. Method according to one of Claims 1 to characterised in that the concentration of Ca 2 ions is between 8 and 30 M.
9. 12. Method according to one of Claims 6 to 11, characterised in that the oocytes are treated with an inhibitor of the expulsion of the polar body, and will constitute a population of diploid parthenogenetic eggs.
10. 13. Method according to Claims 1 and 3 to 11, charac- terised in that, after the step the chromosomes of the oocyte under the second polar body are withdrawn, these oocytes then being used as a recipient for trans- plantation of a nucleus originating from embryos to be cloned.
11. 14. Method according to one of Claims 1 to characterised in that the treated cells are transplanted eggs. Device intended, in particular, for the culture of oocytes, enabling the method according to one of Claims 1 to 14 to be carried out, of the type containing at least one chamber designed to receive oocytes, the said chamber containing at least one fluid inlet and one fluid outlet, characteriscd in that, at the bottom of the chamber, there are one or more orifices whose geometry is such that it opposes the passage of the oocytes, and in that the fluid entering the chamber is withdrawn, at t ileast partially, through the orifices, creating a partial vacuum such that it substantially provides for clumping of the oocytes on the orifice or orifices, a portion of the fluid being discharged by overflow, and in that the J chamber contains two side walls on which electrodes are placed.
12. 16. Device according to Claim 15, characterised in i that the electrodes are parallel, and in that the orifice or orifices are aligned at an equal distance from the electrodes.
13. 17. Device according to one of Claims 15 and 16, characterised in that it contains at least two fluid I 0 0 6 27 inlets.
14. 18. Device according to one of Claims 15 to 17, characterised in that the two fluid inlets are super- posed.
15. 19. Device according to one of Claims 15 to 18, characterised in that the orifice is a single orifice consisting of a rectilinear slit whose width is less than the diameter of the oocytes and whose length enables a large number of oocytes to be treated in the same cham- ber. Device according to one of Claims 15 to 19, characterised in that the chamber can be isolated and placed in an atmosphere of defined and/or sterile com- position.
16. 21. Device according to one of Claims 15 to characterised in that the fluid is admitted to the chamber via a short tube, after passage through a system avoiding the admission of large gas bubbles.
17. 22. Device according to Claim 21, characterised in that the system consists of a glass flask to which the fluid is admitted and discharge piping which comprises a shaft perforated with holes of very small diameter which open in the vicinity of the centre of the flask, the said piping being connected to the tube.
18. 23. Device according to one of Claims 15 to 22, characterised in that the upper inlet permits injection of pulsating medium, and in that the lower inlet is equipped with a suction system permitting suction of a small amount of the pulsating medium during injection of the said pulsating medium.
19. 24. Device according to one of Claims 16 to 23, characterised in that the frequency, duration and inten- sity of the pulses reaching the electrodes are variable and controlled by an electronic system.
20. 25. Device according to one of Claims 15 to 24, characterised in that it enables the alignment of the oocytes and cell fusion to be carried out by the action of a calibrated pulse generated by an electric field.
21. 26. Device according to Claim 25, characterised in r I" 28 that the cell fusion treatment is automatically controlled by an electronic system.
22. 27. A method for the artificial stimulation of an assembly of cells, substantially as hereinbefore described with reference to any one of the Examples.
23. 28. A device for the culture of oocytes, substantially as hereinbefore described with reference to any one of the Examples. Dated 31 October, 1994 Institut National de la Recherche Agronomique Societe Nationale Elf Aquitaine Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 99** 4 *9* 44 49* I r* 4 44( 4 t ft f 1 c c t i i [N:\LIBW]09320:IAR -4 PATENT METHOD FOR THE ARTIFICIAL STIMULATION OF CELLS AND DEVICE FOR THE CULTURE OF OOCYTES. ABSTRACT The present invention relates to a method for the artificial stimulation of an assembly of cells repro- ducing the effect of a biochemical regulation mechanism controlled under natural physiological conditions by an internal oscillator, characterised in that it comprises the following steps: a) the cells are cultured in vitro for a specified time, b) the cells are placed in the pulsating medium after removal of the culture medium, c) the cells are subjected to a pulse generated by an electric field, d) the cells are placed again in the culture medium, e) the above steps are repeated a number of times, and f) cells are obtained in a homogeneous activated state. It also relates to a device for the culture of oocytes enabling the method to be carried out. Si t P PzE>.*, jETOFOTHARIIIL SIUAINO EL