FR2659347A1 - Device for culturing cells which ensures their immobilisation - Google Patents

Abstract

The present invention relates to a cell culture device consisting of a chamber comprising a fluid inlet and a fluid outlet, characterised in that one or more orifices, whose geometry is such that it prevents the passage of cells, exist at the lower part of the chamber, and in that the fluid entering into the chamber is removed at least partially, through the orifices, creating a depression such that it substantially brings about the blocking of the cells upon the orifice(s), a portion of the fluid being evacuated by overflow. This device may comprise, in addition, electrodes on the inner wall of the chamber. The present invention also relates to a process for the artificial stimulation of cells using this device.

Description

 The present invention relates to a cell culture device ensuring their immobilization, allowing their treatment by different media while avoiding their manipulation, and a method of stimulating these cells using this device.

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

 It therefore relates to the procedure for cloning animal embryos and, in particular to obtaining competent recipient oocytes for transplanting a cell nucleus in a homogeneous state.

 The cloning of domestic animal embryos is the way to remedy the genetic variability induced by fertilization and to standardize the genetic improvements of a breed.

 During fertilization, the entry of the sperm will have two main functions - supply of the male haploid genome, - activation of development which restructures the male nucleus by the cytoplasm of the oocyte and promotes nucleus-cytoplasm interactions.

 It takes an average of 12 to 20 hours between fertilization and the first cell division, during which a set of phenomena takes place, the two paternal and maternal genomes having complementary roles for further development of the embryo (Surani et al . 1984, Nature 308, 548-550), but the exact mechanism is unknown.

 The cloning of an embryo is a method which aims to obtain the most living animals following the transfer of the cell nuclei of this embryo (which contains several cells) into enucleated and activated oocytes. The two functions of fertilization are dissociated. In order to obtain further development of the egg, activation of the recipient oocyte must be carried out before the nuclear transplant. Activation can also apply during the first hours of development of the fertilized egg.

 We know that mammalian eggs can be artificially activated by various physical or chemical stimuli, whether electrical, thermal or osmotic shocks, enzymes or anesthetic agents (Kaufman MH 1983 - Early mammalian development parthenogenetic studies - Cambridge University Press). However, activation is always identified with a unique stimulus, limited in time, supposed to mimic the penetration of the sperm into the egg. None of these treatments replicate the series of physiological changes that occur in the egg after penetration of the sperm.

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

With regard to the cloning of embryos, the first undisputed results were obtained by S. Willadsen in 1986 in sheep. They were followed by those of Prather et al in 1987 in cows, and in 1989 in sows. In rabbits, the first individuals from cloning were obtained by Stice and Robl in 1988. The success rates of these experiments reported in the publications do not generally exceed 4%. However, American companies (Granada Genetics, Houston,
Texas) or Canadian (Alta Genetics, Callgary, Alberta), seem to have mastered the cloning procedure in cattle. It is said that a hundred calves have already been obtained on the North American continent. This industrial handling reflects the interest aroused by embryo cloning in cattle. But have the techniques really progressed?
The procedures described in the publications are similar.

The oocytes used underwent an aging period of 6 to 10 hours. This aging is made necessary to promote activation (no conventional activation technique can activate freshly ovulated oocytes even in cattle (Ware et al. 1989). The chromosomes organized on metaphase II are taken "blind" "in the region of the first polar body, but techniques of visualization of the chromosomes by fluorescence are implemented. An embryonic cell coming from a morula is introduced under the pellucid zone and the fusion is obtained by pulses of electric fields.

The activation of the oocyte is usually caused by the cell fusion procedure; in some cases, it is not described. Two researchers were able to obtain a lamb following the transfer into an oocyte of a nucleus originating from an embryonic button cell (Smith & Wilmut, 1989).

 However, the level of results and the complexity of the mechanisms involved, such as the nucleus differentiation stage, the phase of the cell cycle, the state of the recipient oocyte, its age and the activation procedure do not allow us to understand. why some embryos develop and others do not. Many theories for explaining failure such as that based on transcription activity of the transplanted nucleus have been contradicted by some experimental results.

 New data have appeared on the physiological mechanisms triggered by fertilization and in particular with regard to the rhythms of variations in the level of free calcium and second messengers like Inositol (1,6,5) -tri-phosphate (InsP3) (Cuthbertson et al. 1981; Cuthbertson & Cobbold, 1985; Miyazaki et al.

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

 The process is triggered by the sperm within seconds of fertilization. The chain of reactions that leads to the production of InsP3 appears to depend on an influx of calcium ions. InsP3 binds to a specific receptor that controls the activity of a 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 complexation, kinase activation, etc. Calcium is considered to be the main activator of metabolism. This cycle of reactions is reproduced at a frequency of the order of a 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 intracellular signals emitted at a natural frequency, seems to depend on the presence of male and female pronuclei because it could not be observed on parthenogenetic eggs activated by conventional methods (Cuthberston et al. 1981 ; Cuthbertson & cobbold, 1985; Miyazaki, 1988).

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

 The respective genetic contributions of the oocyte and the sperm are well documented in mice. These studies show that the normal term development 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).

 The production of young people by cloning can only be carried out in practice if reliable and reproducible processes are available. The quality of egg activation plays an important role hitherto ignored in development.

 The mastery of activation understood as a rhythmic phenomenon extending over the entire first cell cycle is essential, not only to routinely produce activated and synchronous oocytes but also to allow eggs "cloned" to benefit from this type of imposed activation. to develop normally.

The present invention therefore relates to a method of long-term artificial stimulation of a set of cells reproducing the effect of a biochemical regulatory mechanism, controlled under natural physiological conditions by an internal oscillator, characterized in that it comprises the following steps a) the cells are placed in culture in vitro, for a time
determined, b) washing of the cells with the pulse medium, c) the cells are placed in the pulse medium, d) the cells are subjected to a pulse generated by an electric field, e) the cells are again placed in the culture medium, f) the preceding steps are repeated a number of times, and g) the cells are obtained in a homogeneous activated state.

 This stimulation method, although it can be used in a large number of methods, has been developed within the framework of the activation of oocytes for the cloning of embryos. Recent studies have in fact shown that in mammalian eggs, fertilization is accompanied by a transient increase in the intracellular concentration of free calcium ([Ca +] i], followed by a series of oscillations of this concentration, which lasts at least 4 hours.

 It is not yet known by what means the sperm triggers these variations of [Ca +] i, as well as the exact biological functions of these long-lasting Ca 2+ oscillations. However, they seem to be characteristic of fertilized eggs and have never been observed when the oocytes are artificially activated (, Nliyazaki 1988 - J. Cell. Biol. 106, 345-353).

 The process developed in the present invention will therefore make it possible to periodically stimulate the oocytes by influxes of calcium at the plasma membrane.

 In fact, by introducing Ca 'ions into the pulse medium, they will be able to penetrate the cell by electroporation, that is to say the creation of "pores" generated by the electric pulse.

 Beyond the immediate phenomena caused in the oocyte such as the expulsion of the polar body, the effects of this stimulation will be manifested in the later stages of the development of the embryo, even when the treatment has stopped. This is how the phenomena of compaction and cavitation can be affected.

 This process can be applied to freshly ovulated oocytes obtained by hormonal stimulation of the female. The activation of oocytes under the defined conditions of frequency, intensity and duration causes the expulsion of the second polar body. We can then take the chromosomes which are at the end of the telophase just below the second polar body. We have oocytes in the same physiological state with a pronucleus.

 A blastomer is then introduced under the pellucid zone and cell fusion is carried out by the action of an electric field.

 The method can then be applied to cloned embryos thus obtained, in order to improve their development, that is to say to regularize cell divisions and obtain compaction.

 Stimulation by this process can be applied to freshly ovulated oocytes placed in the presence of cytochalasin B, which inhibits the expulsion of the second polar cell, maintaining a diploid state. We thus obtain a population of parthenogenetic embryos, which can be implanted in recipient females and will present a synchronous development of the fetus and embryonic appendages.

 These experiments, which were first carried out on rabbit oocytes, were confirmed on mouse oocytes, said to be refractory to activation just after ovulation. The performance of the process is therefore reproducible from one species to another. The activation method can certainly be applied to freshly ovulated bovine or ovine oocytes.

 If we subtract the oocytes during treatment, they regress and we obtain certain artefacts, such as metaphase III oocytes reproducible at will, and which offer new possibilities of studies, in particular on the dynamics of the cytoskeleton and activation. of the genome.

 The process was designed based on the effects of electric fields on plasma membranes.

 It has been shown (Zimmermann, 1982) that pulses of electric fields of the order of 1 to 3 kVcm-1 and of duration of a few, can create micropores in the plasma membrane and establish direct communication between the intra and extra cellular media. It was thus possible, for example, to bring calcium into sea urchin oocytes by exposing them to pulses of electric fields in the presence of calcium ions (Rossignol et al 1983). The amplitude of these influxes can be modulated by the duration of the electric field pulse.

 The method involves the combination of an in vitro culture method of a batch of oocytes (or embryos) by perfusion between two electrodes and a washing method with a non-conductive solution containing 0 to 20 pM calcium in which the pulses are delivered.

The presence of a very weakly conductive solution at the time of a pulse allows an electric field to appear between the electrodes. An excessive presence of ions decreases the "electric field" effect.

 The alternation of a culture period and a washing period makes it possible to frequently subject the oocytes to stimulation in a very well defined ionic medium.

 This process can extend to the stimulation of cells by a whole range of simple or complex ionic or molecular signals at variable concentrations which will be determined by the composition of the pulse medium.

 This process makes it possible to modulate the frequency of the signal by the duration between two washes, and its amplitude by the duration of the electrical pulse.

 It is imperative to completely replace the culture medium with the pulse solution, since the intensity of the current due to excess ions from the culture medium would destroy the oocytes. Immediately after the pulse, the pulse solution is replaced by the culture medium, as the oocytes cannot survive in the pulse solution.

 This process can be controlled by means of software which will make it possible to create stimulation treatments according to specific, exponential, sinusoidal, Fourier sequence or other equations.

 One of the aspects of the present invention is the simultaneous treatment of a set of oocytes, which will thus be obtained, activated in the same physiological state. The core transplants can thus be performed on physiologically identical oocytes.

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

 It is extremely interesting to be able to standardize the different phases of oocyte processing, in order to obtain good reproducibility of the process.

 The present invention therefore relates to a dynamic system, operating continuously and which can be automated, produced by a device allowing the automatic succession of the washing and stimulation stages and the acquisition of the stimulation parameters.

 This device is characterized by the fact that it immobilizes the cells during the different phases of the treatment.

 I1 consists of an enclosure comprising a fluid inlet and a fluid outlet and, is characterized in that at the lower part of the enclosure are one or more orifices whose geometry is such that it opposes at the passage of the cells and in that the liquid entering the enclosure is withdrawn, at least in part, through the orifices, creating a vacuum such that it substantially ensures the blocking of the cells on the orifice (s), a part fluid being discharged by overflow.

 This device makes it possible to retain the cells without damaging them or introducing parasitic mechanical stress. In addition, it allows cells to be replaced, removed or moved at any time. This device therefore makes it possible to successively place the cells in media of chemical composition of different natures, for periods and at a variable frequency.

 The enclosure preferably comprises on its internal walls two parallel electrodes, and the cells are aligned at equal distance from the electrodes, immersed in the medium corresponding to the phase of the treatment in progress.

 Thanks to this device, it is possible to cultivate the cells at will or else into the impulse medium to penetrate into the treated cell, an ion, a molecule, a complex chemical or biological substance.

 These media, both the culture medium and the pulse medium, are in continuous circulation, and it is this flow of liquid which by suction effect ensures the maintenance of the oocytes between the electrodes during the renewal of the medium.

 The compositions of the culture media are known and depend on the cells in culture. The pulse medium generally consists of a nonionic medium to ensure a field effect. It will be an isotonic solution, for example of glucose.

 The presence of a large amount of ions can seriously damage cells during the pulse. It is therefore necessary to wash the cells with the pulse medium to remove the ions contained in the culture medium.

 This device can be designed as an activation chamber, the capacity of which can be adapted to the number of cells which it is desired to treat simultaneously.

 The characteristics of the process using the device. can vary widely and are generally only limited by conditions such as wash times. Thus, the frequency of the pulses is limited by the minimum washing time by the pulse medium.

 The parameters of the process and of the device, that is to say the inflows and outflows of fluid, as well as the frequency, the duration and the intensity of the electrical pulses arriving in the electrodes are managed by an electronic system.

 The characteristics of the electrical impulses in themselves can bet and depend on the cells and the aims sought.

 In general, the electric fields vary from I to 3 Kv. cm 1 and the electric pulse has a duration of 10 us to 2000 lls.

 The organization of the tubes which open into the chamber allows media to be injected without the oocytes being taken down by the gas bubbles which eventually form in the pipes. In addition, the distribution structure of the media makes it possible to considerably limit the ionic contamination of the stimulation medium by the ions of the culture medium.

 The media injection pumps can be controlled by a microcomputer which will determine the repeatable washing sequences while keeping the cells between the electrodes.

 This device can be applied to the treatment of cells with any substance which it is desired to make penetrate into the intracellular space. The composition of the pulse medium will determine the penetration of this substance according to a concentration gradient.

 During a cell culture, it will be possible to cause the desired substance to enter all of the cells at a given time, which may be unique. For this, the culture medium will be replaced by the pulse medium and the electric field pulse will be triggered, the duration of the pulse determining the quantity of substance which penetrates. This pulse medium can then be removed and the cells returned to culture in an appropriate medium. The advantage of this system is that it allows simultaneous processing of a set of cells which are thus synchronized. It operates continuously and avoids any manipulation of the cells, thus ensuring better reproducibility and greater speed of execution. This allows standardization of conditions and facilitates technology transfer.

 It is conceivable to make an ion or a simple molecule enter cells. This device can also be applied to the stimulation of cells by a more complex molecule, or to the penetration of DNA fragments, or of an episome.

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

 This device enables the activation and synchronization process of oocytes to be carried out by repeated Ca stimulations for the cloning of domestic animal embryos.

 An embodiment of the chamber is shown in FIG. 1.

 FIG. 2 represents the pulse duration curves for the different treatments applied to the oocytes.

 In FIG. 1, the oocytes 1 are placed on a rectilinear slot 4, produced by the juxtaposition of two glass plates 3 and 5, the spacing of which is less than the diameter of the oocytes. The media, culture medium and pulse medium, arrive in the upper part of the chamber each through a separate tube, respectively 10 and 11, avoiding reciprocal contamination.

 The medium 9 corresponding to the current treatment phase arrives continuously and is discharged through a tube 6 located at a lower level than that of the oocytes. This current of liquid keeps the oocytes pressed against the bottom of the chamber by suction.

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

 The enclosure 8 is thermostatically controlled at 38 "C.

 Each medium arrives in the chamber after passing through a system retaining the large gas bubbles.

This system consists of a glass balloon into which the liquid arrives. From this balloon leaves a gold rod perforated with small diameter holes: after passing through this rod, the gas bubbles of size
Important are retained and the liquid arrives in the room by a short tube.

Rabbits were superovulated by injection of FSH and LI, and the oocytes were taken from the oviducts. After treatment 5 min by
hyaluronidase, the oocytes are placed in the activation chamber, in medium B2 (menez, 1976 - C. Hebd. Seanc. Acad. Sci. Paris 282,
1967-1970) at 38 C, in an atmosphere with 5% CO2.

 Oocytes can be immediately subjected to the activation treatment, no aging period is necessary.

They are subjected for 90 minutes to a series of pulses of electric field, at a rate of one pulse every 4 minutes, that is to say 22 pulses of decreasing duration dissipating a total energy 1250 ujoules, of a total duration of 14 868 ps , the electric field has a value of 1.8
Kv. cm-1.

 Before each pulse, the culture medium is replaced by a pulse medium consisting of an isotonic solution of glucose containing CaCl 2 10 pu.

 At the end of the activation treatment, all oocytes have two well-formed polar cells. A homogeneous population of activated oocytes in the same physiological state is therefore obtained at the same time, within a few minutes. This has two advantages: the first is to always be able to perform core transplants in physiologically identical eggs, the second, more practical, is to be able to operate when the chromosomes (haplold) are all at the end of telophase just below the second polar body. This natural location of the place where the chromosomes are located facilitates their "blind" removal and allows, without resorting to fluorescent labeling techniques, to quickly chain the manipulation steps.

 The chromosomes can then be removed. We notice that the two polar globules are not always joined (some are opposite one another, this is why the first polar globule is not a good marker of the place where the maternal chromosomes). The effectiveness of this operation, verified by the subsequent presence of a pronucleus, is greater than 80%. A blastomer is then introduced under the skin area. Fifteen to 20 oocytes can be handled in 1 hour.

 The cell fusion is then carried out. The cell fusion method derives from Zimmerman's work 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 automatically, under the control of a software, the alignment and the cell fusion in the stimulation chamber. This procedure will limit the duration of the manipulations and standardize the experimental conditions.

MATERIALS AND METHODS
Superovulation was induced in sexually mature female rabbits of various species by subcutaneous injection of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in accordance with the technique described by Kennely and Foote (1965 ) and modified by Thibault (personal communication). Females received 2 mg of
FSH in five injections 12 hours apart: 0.250, 0.250, 0.650, 0.650 and 0.255 mg. 12 hours later, before the protrusion by a vasectomized male, they were injected with ru, 33 mg of LH.

 The oocytes were recovered from the oviducts 12-15 h after the projection by washing with a buffered phosphate saline solution (PBS). They were incubated for 5 minutes in hyaluronidase (300 l.u.ml-I in PBS) to remove the follicular cells. After the treatment, the oocytes were cultured at 38 in B2 medium (Ménézo 1976) in an atmosphere at 5 Ó of CO2.

Experimental method and procedure
The membrane permeability of freshly ovulated rabbit oocytes was transiently increased by opening the pores with
2+ a pulse induced by an electric field in the presence of Ca 10 PM contained in a 0.3 M glucose solution - 18 MOhm H2O (pulse medium). It is accepted that. while the pores are open, an ion flow circulates according to the concentration gradients through the cell membrane towards the interior of the cytosol as has already been shown in sea urchin oocytes (Rossignol et al, 1983). Thus, the ion influx can be adjusted under these conditions either by the difference in ion concentration between the interior and the exterior of the cell or by the duration of the pulses.

 The experimental procedure for an electrically induced ion flux was similar to that previously described for the electric field fusion of two-cell rabbit embryos (Ozil and Modlinski 1986). Oocytes are cultured with M16 medium (Whittingham, 1971) at 380C in a specially designed chamber. Before each pulse, the culture medium is automatically replaced by the pulse medium.

The details of the chamber are described in Figure 1. Each pulse was composed of two alternative pulses in order to avoid "lateral electrophoresis" (Jaffe 1977) of the membrane proteins which could occur after several pulses of the same polarity (Poo, nineteen eighty one). The amplitude of the ion signal was modulated over the duration of the pulse. The entire process was controlled by a microcomputer
IBM PC-AT 286 via a Tektronix MI 5010 interface with a program written in MS-BASIC. The actual voltage and current between the electrodes were measured by a Tektronix 77041 oscilloscope mounted with a programmable digital converter 7D20 and an amplifier 7A22.

 The rhythm of the electrical pulses and the total duration of the treatment were the same for all the treatments, that is to say applying a pulse every four minutes for 90 minutes.

These x values were chosen because they agree well with the frequency and the average duration of hyperpolarization of the membrane potential of rabbit oocytes during fertilization (22 biphasic oscillations of membrane potential during the first 90 minutes after sperm-egg fusion, one pulse every 4 minutes -McCulloh et al., 1983). We know that the variation in membrane potential reflects the passage of K based on calcium activated channels and that they therefore constitute a reliable indicator of [Ca2 Xi (Miyazaki and Igusa 1982).

The amplitude of electric fields (1.8 kVcm-l) was constant for all the experimental groups.

Oocyte treatment
The effects of the different treatment parameters on the activation of oocytes 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 culture medium and electrical pulses), freshly ovulated oocytes and fertilized eggs subjected to 22 double pulses of 900 us in the pulse medium devoid of Ca2 + (the first impulse is given 13 to 15 hours after the projection). After treatment, the fertilized eggs were transferred to recipients to determine term survival. Unfertilized oocytes were cultured in vitro and the rate of parthenogenetic activation was noted.

Group B - calcium ions and pulse duration
The effect of the pulse duration was studied in the pulse medium containing 10 pM CaCl2. The treatment for which the oocytes are not activated was considered to be the minimum duration and that resulting in the lysis of the oocytes 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, 1200 and 1500 lls respectively. The effect of the presence of Na and Mg ions at a concentration of 10 M in the pulse medium was tested with 22 constant double pulses with a duration of 900 ps.

Group C - pulse modulation and type of parthenogenetic activation
Processing 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 activation rate with a uniform type of parthenogenetic egg. In order to check whether a progressive reduction of calcium stimuli in a given treatment has an effect on the type of parthenogenetic reaction, the oocytes were subjected to treatments in which the duration of the pulses gradually decreased according to a negative exponential relationship. Four treatments were tested in accordance with the maximum duration of the first pulse. FIG. 2 gives the curve of the pulse durations for these treatments.

 A negative exponential relation (D (n) = e (axn + b) + c) between the pulse index (I to 22) and the pulse duration (1500 us at 200 ps) was arbitrarily chosen for find the value of each pulse during processing.

 With D (n): cycle pulse duration (n).

 The coefficient a determines the slope of decrease of this relation.

 The coefficient b determines the duration of the first pulse.

 The coefficient c determines the duration of the last pulse.

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

b = 5.9920 for treatment 1
= 6 5510 for treatment II
6.9080 for treatment III
7.1700 for IV treatment.

Group D - Electric field modulation and in vitro development
The eggs are treated in the presence of 8 μg-l of cytochalasin B in the culture medium to block the expulsion of the second polar globule and to obtain a uniform population of diploid parthenogenetic eggs. Two treatments were applied to group C, treatment I which is the weak treatment with a total pulse duration equal to Il 228 us and treatment III which is the strong treatment with a duration of 14 868uns. Parthenogenetic eggs were cultured in vitro up to the blastocyst stage and the influence of the two treatments was evaluated by the rate of blastocyst formation.

Viability beyond the implantation of diploid parthenogenetic embryos
The 4-cell stage embryos were transplanted into recipient oviducts. The fertilized eggs were also transplanted into the opposite horn of a few recipients in order to compare parthenogenetic development with normal development. The recipients were autopsied between day 8.5 and day 13 of gestation and the number of implantation sites for living fetuses was noted.

RESULTS
Group A - Effect of the experimental environment (controls)
When the pulse medium does not contain electrolytes, none of the oocytes (105) are activated. The experimental environment and the culture conditions, that is to say the replacement of the culture medium before each pulse with a pulse medium containing no electrolytes and the treatment with relatively strong electrical pulses (2 x 1.8 kVcm-1x900Ps x 22 times, i.e. a total pulse duration of 30,600, us) has no visible effect on freshly ovulated oocytes but the conditions do not allow development to be triggered. On the other hand 41% (9/22) of the fertilized eggs treated in the same way developed in the long term demonstrating thus that the treatment by high voltage electric impulses does not have an opposite effect on the development capacity of the fertilized oocytes . It was not possible to completely replace the culture medium with the pulse medium before each pulse. The current measured during the pulse reveals that the conductivity of the pulse medium is at least 15.6% higher than that of the pulse medium measured between the electrodes before the experiment and before the first injection of culture medium.

During the experiment, ions from the culture medium are still present during the pulses and this modifies the ion signal.

These variations in the microenvironment do not appear to have any significant effect on activation or embryonic development. In this series of experiments, the eggs are washed with the pulse medium for 45 seconds every four minutes. This period during which the eggs are not in the culture medium has no significant effect on activation or on long-term survival.

Group B - calcium ions and pulse duration
Table I summarizes the results of the experiment in which the activation rate was tested in relation to the pulse duration in the presence of 1M CaCl 2.

 The appearance of pronuclei after 3 or 4 hours of culture serves as a marker for parthenogenetic activation. These results clearly show that parthenogenetic activation is triggered when the pulse medium contains Ca 2+ 10 I1M.

 In addition, the activation rate is directly related to the duration of the pulse which indirectly controls stimulation by calcium. The duration of ionic stimuli influences not only the activation rate but also the nuclear configuration of 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 - pulse modulation and type of parthenogenetic activation.

 The results are summarized 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 (91%) of them have a single pronucleus and two polar cells when the meiosis is over.

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

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

Group D - Effect of electric field modulation on in vitro development of oocytes activated by parthenogenesis.

 The results are summarized in Table III. No visible difference in the development of each group could be noted until the third division.

 In the parthenogenetic embryos produced by treatment I, a smaller proportion of embryos exhibit compaction and those that compact are irregular. On the other hand, the majority of embryos produced by treatment III compacts and develops until the blastocyst stage.

 These results show that the shape of the activation stimulus has a profound effect on the ability of parthenogenetic embryos to develop up to the blastocyst stage in vitro.

Post-implantation viability of diploid parthenogenetic eggs.

 13 recipients became pregnant and were autopsied between day 8.5 and day 13. The results are summarized in Table IV.

Although the parthenogenetic embryos are smaller than the control embryos, they appear morphologically normal according to the criteria defined for the rabbit by Edwards (1968). The ratio between the size of the embryonic appendages and that of the fetus is roughly equivalent for embryos obtained by fertilization or parthenogenesis.

 Thus, it seems that the general development of parthenogenetic embryos, the fetus and its trophoblastic tissues is slowed down.

 Dead fetuses have several anomalies and it was not possible to classify them.

Table 1
Effect of the presence of calcium ions and duration of the electrical pulse on the activation of rabbit oocytes
Duration Number Number%% of activated oocytes with pulse of lysed oocytes (%) activated 1 PN + PB1 2PN 2PN # (us) & B2 + PB 1 + PB 1
200 us 47 0 (0) 13 83 17 0
300 s 99 0 (0) 62 68 25 7
600 s 85 0 (0) 98 71 19 10
900 s 63 0 (0) 100 62 14 24 1 200 s 97 5 (5) 100 57 29 14 1 500 ps 50 3 (6) 100 30 17 53
Table 2 - Effect of modulation of the electric field on activation
Treatment Number ~~% oocytes activated with
oocyte activation 1 PN + PB1 2PN 2PN #
& B2 + PB1 + PB1
I 86 88 83 9 8
II 151 99 89 9 1
III 118 100 91 9 0
IV 96 100 70 25 5 # Abnormal pronuclei, small micronuclei
Table 3
Effect of electric field modulation on the development of parthenogenetic eggs in vitro
Treatment Number% Number Number Number
of cultured oocyte activation morulae blastocysts
compact (%) (%)
I 98 91 69 25 (36) 23 (33)
III 352 100 244 244 (100) 216 (88)
Table 4
Post-implantation development of parthenogenetic eggs subjected to treatment III.

Day of autopsy D 8-9 D 9-10 D 10-11 D 12-13 Total
Number of eggs transferred 21 26 50 68 165
Number of locations 9 3 15 23 50% cumulative (42.8) (21.2) (27.8) (30.3)
Number of living fetuses 8 3 7 0 18% cumulative (88.8) (91.6) (66.6) (36)
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Cuthbertson, K.S.R. Whittingham, D.G. & Cobbold, P.H. (1981). Free Ca2 + increases in exponential phases during mouse oocyte activation. Nature 294, 754-757.

Cuthbertson, KSR & Cobbold, PH (1985). Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2 +,
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Cuthbertson, K.S.R. (1983). Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol. J. Exp. Zool. 226, 311-314.

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Claims (20)

 1. Cell culture device consisting of an enclosure comprising a fluid inlet and a fluid outlet, characterized in that at the bottom of the enclosure, there are one or more orifices the geometry of which is such that it opposes the passage of cells and in that the fluid entering the enclosure is withdrawn, at least in part, through the orifices, creating a vacuum such that it substantially ensures the blocking of the cells on the orifices, part of the fluid being discharged by overflow.  2. Device according to claim 1, characterized in that electrodes are placed on the internal wall of the enclosure.  3. Device according to claims 1 and 2, characterized in that the enclosure has two side walls on which the electrodes are placed, the orifice or orifices being aligned at equal distance from the electrodes.  4. Device according to claims 1 to 3, characterized in that the orifice is a single orifice, constituted by a rectilinear slot whose width is less than the diameter of the cells.  5. Device according to claims 1 to 4, characterized in that the slot is created by the juxtaposition of two glass plates.  6. Device according to claims 1 to 5, characterized in that the enclosure can be isolated and placed in an atmosphere of defined composition and / or sterile.  7. Device according to claims 1 to 6, characterized in that the fluid inlets and outlets are managed by an electronic system.  8. Device according to claims 2 to 7, characterized in that the frequency, duration and intensity of the electrical pulses arriving in the electrodes are managed by an electronic system.  9. Device according to claims 1 to 8, characterized in that the fluid arrives in the enclosure by a short tube, after passage through a system preventing the arrival of gas bubbles of large size.  10. Device according to claim 9, characterized in that the system consists of a glass balloon into which the fluid arrives and of a discharge pipe which consists of a tube perforated with very small diameter holes which open in the vicinity of the center of the balloon, said piping connecting to the short tubing.  11. Device according to claims 1 to 10, characterized in that fluids of different compositions can arrive successively in the enclosure, and that the systems of arrival and extraction of fluids avoid their mutual contamination.  12. Device according to claims 2 to 11, characterized in that - the fluid present in the enclosure is a pulse medium, containing at  minus a substance ensuring the appearance of an electric field and  possibly a molecule or any specific substance we want  penetrate the cell, and - an electric field pulse is applied via the  electrodes.  13. Device according to claims 1 to 12, characterized in that the electrical system allows the sequence comprising the following steps to be carried out on the cells - culturing the cells in an appropriate culture medium for  a determined time, - replacement of the culture medium by an impulse medium, - sending of an electric field impulse via the  electrodes, - replacement of the pulse medium by a culture medium.  14. Device according to claims 1 to 13, characterized in that the treated cells are oocytes.  15. Device according to claims 1 to 13, characterized in that the treated cells are fertilized eggs.  16. Device according to claims 1 to 15, characterized in that 2+ that the pulse medium contains Ca  17. Device according to claims 1 to 16, characterized in that the pulse medium contains Ca at a concentration of 10 PM.  18. Device according to claims 1 to 17, characterized in that the sequence to which the cells are subjected is repeated several times, with a frequency which can vary, the duration of the electric field pulse being able to be different during each sequence .  19. Device according to claim 18, characterized in that it makes it possible to achieve cell alignment and cell fusion by the action of a calibrated pulse generated by an electric field.  20. Device according to claim 19, characterized in that the cell fusion processing is automatically managed by an electronic system.