WO1998003633A1

WO1998003633A1 - Enzymatic process for the dissociation of animal tissue cells and application of such process

Info

Publication number: WO1998003633A1
Application number: PCT/ES1997/000169
Authority: WO
Inventors: Carmen Prada Elena; Rosario Lopez Lopez; Meritxell Lopez Gallardo; Agustin Prieto Prieto
Original assignee: Universidad Complutense De Madrid
Priority date: 1996-07-24
Filing date: 1997-07-04
Publication date: 1998-01-29
Also published as: ES2111497A1; AU3263797A; ES2111497B1

Abstract

The invention relates to a new process of disgregation of animal tissues, and specially of those of the CNS of vertebrae, which provides for the separation of viable cells, while preserving their native morphology, through the use of proteases of Streptomyces fradiae and in specific conditions (low ionic strength, osmolality between 175 and 300 mOsm/kg, pH between 6.2 and 7.4 and temperature between 32 and 36 °C) in the disgregation process. Said results which cannot be obtained with prior disgregation processes, represent an important improvement for applications such as a) the use of dissociated cells of the embryonic CNS in cell cultures for growth studies and pharmacological products, and neuronal transplants; b) use of neurones and glial cells dissociated from adult CNS in electrophysiological studies and fine location of molecules in subcellular structures; and c) purification of specific populations of cells for biochemical and genetic studies.

Description

TITLE OF THE INVENTION

Enzymatic procedure for the dissociation of cells from animal tissues and their application.

OBJECT OF 1 - INVENTION:

The present invention falls within the technical field of Biotechnology. More specifically, the invention consists of a new method for the dissociation of cells from the tissues of the central nervous system of vertebrates using Streptomyces fradiae proteases. The main advantage of this method compared to those currently used for the same purpose, is that it dissociates cells while preserving their native form and structure. Because of their superior state of conservation, the cells thus dissociated are more suitable than those obtained by other known methods, for important applications in biomedicine, such as cell culture, cell transplantation, new cell and molecular biology techniques, and others.

BACKGROUND

Tissue dissociation is the first step in the performance of cell cultures. Cells can be dissociated by strictly mechanical, chemical or enzymatic methods. In enzymatic procedures, the use of proteolytic enzymes is usually combined with mechanical procedures

Virtually all proteases used in the disintegration of animal tissues are raw commercial products that contain mixtures of proteases with different activities, and may also contain lipases, glycosidases and nucleases (Waymouth, C. To disaggregate or not to disaggregate. Injury and cell disaggregation. , transient or permanent ?. In vitro, 1974, 10 97-111), (Waymouth, C Methods for obtaining cells in suspensions from animal tissues, In. Pretlow, Th. G. and Pretlow, Th. P. (Eds.) , Cell separation methods and selected applications. 1982, Vol 1, Academic Press, Inc. New York, pp. 1-29) Therefore, the enzymes used so far are not specific to the extracellular matrix, but also act on proteins, lipids and carbohydrates of the plasma membrane, always causing damage to cells, to a greater or lesser extent (Waymouth, O In vitro, 1974,10 97-111) Despite this lack of specificity on extracellular matnz, some proteases are more appropriate than others to dissociate certain tissues, and special applications have been developed for some of them. Thus, certain collagenases are selectively used to disintegrate the liver (Seglen, PO Preparation of isolated rat liver cells Methods Cell Biol, 1976, 13. 29-83) or pancreatic islets (Lacy, P E. and Kostianovsky, M Method for the isolation of intact islets of Langerhans from the rat pancreas Diabetes, 1967, 16 35-39), dispasa is the enzyme of choice in the "in vitro" preparation of human epidermis laminae, used for transplants (Green, M., Kehmde , O and Thomas, J Growth of cultured human epidemial cells mto multiple epithelia suitable for grafting Proc Nat l Acad Sci. USA, 1979, 76 5665-5668) and elastase is the appropriate enzyme to dissociate lung cells or to digest elastin fibers of the arteries (Waymouth, C 1982, cit work) Trypsin is undoubtedly the most commonly used enzyme for the disintegration of the central nervous system (CNS) of vertebrates (Freshney, RI Culture of animal cells A Manual of Basic Technique, Alan R Liss , Inc, New York 1987), (Banker, G and Goslm, K Pπmary dissoαated cell cultures of neural tissue In Banker, G and Goslm, K (Eds), Cultuπng nerve cells, MIT Press, Cambπdge, Mass, 1991, pp 41 -73), although papain, collagenase, pronase, or dispase have also been used to disintegrate the hippocampus or retina, either alone or in admixture with other enzymes. Raw tπpsma is the widely used enzyme for dissociation of embutonic CNS. This protease is a multienzyme complex that, in addition to trypsin, may contain chymotrypsin, collagenase, πbonuclease elastase, phosphatase, lipase and amylase (Waymouth, C 1982, cit work) The same trypsin breaks down peptide bonds of which lysine or arginma are part , amino acids present in any protein (Waymouth, C. 1982, cit.). For this reason, the cells are always damaged (Waymouth, O 1974, cit work), and the result of the dissociation are rounded cells, devoid of their prolongations and morphologically non-identifiable, although many of them retain the ability to divide, grow and differentiate, at least to some extent, in a culture medium (Freshney, RI 1987, work αt) and (Banker G and Goslm, K. 1991, work αt). Since the embryonic and adult CNS cells of vertebrates, both neurons and glial cells, are morphologically and structurally complex, and have subcellular domains in which specific functions are performed, some already well characterized, the isolation of cells without altering its morphology and preserving its functional integrity has been a permanent challenge for neuroscientists. Some neurons of the adult CNS have been isolated with some degree of integrity, sufficient to study the electrophysiology of their membranes, introducing minor variations in the methods used for disintegration of the embryonic CNS (Bader, CR, MacLeish, PR and Schwartz, EAJPhysiol. (Lond .), 1979, 296 1-26), (Sarthy, PV and Lam, DMK Brain Res., 1979, 176: 208-212), (Tachibana, M, J Physιol., 1981, 321: 141-161), (Newman, E. A, J. Neurosa., 1985, 5 2225-2239), (Kay, AR and Wong, R K. S, J. Neurosci. Methods, 1986, 16: 227-238), (Kaneda, M., Nakamura, H. and Akaike, N. Neurosa. Res., 1988, 5: 299-315), (Reichenbach, A., Wolburg, H., Ríchter, W. and Eberhardt, W., J. Neurosci Methods, 1990, 32 227-233), (Chad, JE, Stanford, I., Wheal, HV, Williamson, R and Woodhall, G, In Chad, J and Wheal, H (Eds.), Cellular Neurobiology A Practical Approach, IRL Press Oxford, 1991, pp. 19-37), (Sayer, RJ, Brown, AM, Schwmdt, PC and Cπll WE, J Neuroρhysιol., 1993, 69: 1596-1606), (Tumer, RW, Borg, LL and Syed, NI, CNS J Neurosci. Methods, 1995, 56: 57-70) among others. However, the embryonated CNS cells obtained by dissociation with any protease, using current methods, are always rounded (Banker, G. and Goslin, K. 1991, work αt), (Adler, R Nature and nurture in the differentíatjon of retinal photoreceptors and neurons. Cell Dιffer, 1987, 20 183-188), because only their somas get rid of the damage caused by these enzymes

There is no standard enzymatic method for tissue dissociation, although any of the published methods includes a number of common stages, such as shredding and / or shredding of the tissue, incubation with dissolved proteases in simple isotonic saline phosphate buffer or in salt solutions. balanced lacks Ca ⁺² and Mg ⁺² , and inhibition of enzymatic action There are many described vaπables that can modify the result of a tissue dissociation, among which are the degree of cleavage of the tissue, osmolality and pH of the solutions used, the time and temperature of incubation of the tissue with the protease solution (s), the presence of nutrients, such as glucose, in the solutions, the way in which the tissue fragments are crushed during or after the incubation (Waymouth, O 1974, work cit.), (Bashor, MM. Dispersion and disruption of tissues. In ^* Jakoby, WB and Pastan, IH (Eds.), Methods in enzymology, Vol LVIII, Academic Press, Inc., New York, 1979, pp. 119-131), (Waymouth, 1982, cit work), (Kay AR and Wong, R KS, 1986, cit work), (Kaneda, M., Nakamura, H. and Akaike, N, 1988, cit work), (Banker, G and Goslin, K.

1991, cit work.)

EXPLANATION OF THE INVENTION

The present invention relates to a method of cell dissociation, whereby live cells of the embryonic and adult CNS of vertebrates are obtained with their preserved native forms and structures

The procedure is mainly characterized by the use of a complex of alkaline proteases produced by Streptomyces fradiae, which we will refer to hereinafter as "SF-protease", never before used to dissociate cells from animal tissues, and from a dissociation medium of a different composition than those used in known methods.

The procedure serves for the disintegration of animal tissues, such as the CNS tissues of chicken, rat and rabbit, and other tissues of these and other species. Protease-SF differs from the proteases previously used for the breakdown of the CNS in that it is free of lipases, nucleases, amylases and glycosidases, which makes it less harmful to cells than commercial proteases.

Protease-SF is obtained by fermentation with a strain of Streptomyces fradiae, preferably Streptomyces fradiae ATCC 10745 or Streptomyces fradiae ATCC 14544, in an aqueous medium containing, in g / l, 30.0 starch, 10.0 flour soybean, 5.0 "corn steep solids", 1.0 dipotassium phosphate, 0.25 magnesium sulfate, 0.01 ferrous sulfate and 10.0 calcium carbonate Fermentation is aerobic, in submerged culture, at 28-35 ° C and with a duration of 24-72 h Next, the proteases present in the liquid phase of the fermented broth are concentrated and purified by filtration or centrifugation thereof, precipitation with ammonium sulfate, chromatography and precipitation in aqueous acetone solutions, until reaching a solid protease-SF preparation with the following characteristics: a) A specific proteolytic activity of 20,000-25,000 UAP / mg, the unit of proteolytic activity (UAP) being defined as the amount of protease-SF that, acting on heme Denatured oglobin, releases, in ten minutes, at pH 7.5, 25 ° C, hydrolysis products with a total absorbance, at 280 μm, equal to that of 1 μg of tyrosine. b) Hydrolytic activity on animal and vegetable proteins (hemoglobin, casein, collagen, gelatin, elastin, keratin, gluten, gliadma, etc.). c) Proteolytic activity at pH 5-12 d) Proteolytic activity not activated by compounds with sulfidyl groups or inhibited by compounds that block said groups, but slightly inhibited by tnpsma inhibitors from soybeans and pancreas, and strongly inhibited by very low concentrations of isopropylfluorophosphate, typical behavior of serine proteases e) Absence of contamination by glycosidases amylases, lipases and nucleases f) Great stability in solid state at room temperature Solution stability is much lower than in solid state but increases considerably in presence of calcium ions and, under these conditions, it is maximum at pH 8.2 During the development of the invention different samples of SF-protease were used with activities of 1,700-35,000 UAP / mg However, as different samples of protease- SF may give different results the procedure object of this invention has been established using samples Protease-SF of 20,000-25,000 UAP / mg

Unlike all the commercially available proteases for tissue breakdown, which are used at concentrations in volume at 1 mg / ml, the optimal concentration of SF-protease to disintegrate the tissues of the central nervous system varies between 4 and 156 μg / ml, depending on the animal species, type of tissue and stage of development or age of the animal The optimal concentration is defined here as that which allows obtaining the greatest number of viable cells and with their native forms, of one type or of all types that make up the tissue that is the object of disintegration, using the procedure described below

For the dissection and slicing processes of the tissue, and for the preparation of the incubation medium, a means of dissociation of osmolality is used between 175 and 300 mOsm / kg, depending on the type of tissue unlike the dissociation means used in published protocols that are all isoosmotic with plasma, having an osmolahdad around 300 mOsm / kg The appropriate osmolality for each type of tissue is indicated in table 1 below. The composition of this medium is sucrose 175- 300 mM in 1mM sodium phosphate buffer or in 5 mM HEPES buffer, at pH 6.2-7.4, or simply in distilled water, pH 6.4-6.5 Therefore, this medium also differs from the means used in the disintegration procedures published for having low ionic strength, which is a necessary condition for the proper functioning of the SF-protease. Regarding the pH of the dtso aonon medium, the results do not vary appreciably in the range 6.2- 7.4 The incubation medium consists of a protease solution in the dissociation medium defined in the previous paragraph, at the appropriate protease concentration for each type of tissue, animal species and stage of development as also indicated in Table 1 Since the Protease activity in the dissociation solution decays progressively over time in the temperature range of 4 to 25 ° C, and is lost by freezing the solution, it is necessary to prepare the incubation solution immediately before starting tissue dissection

Table 1. Optimum concentrations of the SF-protease to dissociate viable and well-preserved morphologically and structurally preserved cells from different chicken and rat CNS tissues.

Tissue Stage N "dc Osmolalulad Range Cell types experiments medium IIK IIII.HKHI do concentration

Retina E8-E18 142 | 7 ^ - ^* ! Ι ιu ^* tl-76 Neuroblasts and neurons of all types, Müller cells (glia) in differentiation

E1 -adult 92 76- 152 All types of neurons, with the exception of gang onar cells, Müller cells

Cerebellum E12-E19 55 17S-1I K I 46-156 Purkinje cells at different stages of differentiation and morphologically differentiated, other cell types

RAT

Retina PO-adduct 56 15-26 Neurons in different stages of differentiation and differentiated of all types, Müllcr cells in differentiation and adults

Cerebellum PO-Pl l 29 4-73 Pur inje cells at different stages of differentiation and morphologically differentiated, other cell types

Hippocampus PO-Pl l 91 4- 17 Pyramidal cells at different stages of differentiation and morphologically differentiated, other types of neurons, gha cells

SUBSTITUTE SHEET (RULE 26) Description of the procedure object of the patent.

The procedure is specified in four essential stages common to the multiple variants thereof, which must be used to disintegrate the different tissues of the central nervous system of the different animal species. 1 ": Chopping tissue.

It is carried out in a volume of dissociation medium approximately twice the volume of the tissue, sufficient to perform the finest manual slicing at a temperature of 20-23 ° C. The osmolality of the dissociation solution, from 175 to 300 mOsm / kg, depends on the type of tissue as previously indicated (Table 1) 2 ^a : Placement of the tissue in the incubation medium

The tissue is passed with the dissociation medium in which it has been cut into a tube containing the incubation solution at a temperature of 28-36 ° C, at this time the enzymatic digestion begins. The concentration of the protease-SF in the incubation medium, from 4 to 156 μg / ml, depends on the animal species, the type of tissue and stage of embryonic development or age of the animal, as previously indicated in Table 1 .

The volume of incubation medium is in relation to the mass of tissue to be dissociated. The tissue mass / volume ratio of the incubation medium is a variable that clearly influences the result of the dissociation. The magnitudes of the two terms of the relationship are determined experimentally based on the harvest of cells to be obtained, the type of tissue and the stage of development or age of the animal. For guidance, the mass / volume ratios that provide the maximum number of morphologically well-preserved cells are a 3-day postclosion chicken retina (P3) in 600 μl of incubation solution, a 3-day rat cerebellum gyrus (postnatal) P3) in 800μl, or four hippocampus of rat P3 in 800μl. Since the harvest of cells (number of cells per portion of tissue, or by volume of the medium containing the cell suspension at the end of the dissociation) depends not only on the tissue mass / volume ratio of the incubation medium, but also on the degree of tissue chopping and mechanical support during the enzymatic digestion process, the tissue mass / volume ratio of the incubation medium is adjusted based on these two variables, which in turn are set and controlled for reproducible results. As an example, the ratio of a P3 chicken retina in 600 μl is suitable to obtain 6.6-8.3 x 10 ⁵ cells / ml (40-50 x 10 ⁶ cells / retina), if a chopping is done very fine tissue and is helped mechanically by carefully passing the cell suspension ten times through a 1 ml micropipette tip, at the beginning of the incubation and every 10 minutes in a 45 minute incubation at 35 ° C. 3 ^a : Tissue incubation.

The tube containing the chopped tissue is placed in the volume of incubation medium, at the proper ratio, in a tube holder that is placed in a constant temperature water bath.

The incubation temperature can vary between 32 and 36 ° C, depending on the type of tissue, stage of embryonic development or age of the animal. The tissues of young embryonic stages disintegrate at a lower temperature than those of advanced stages, and at a lower temperature the cellular damage is lower and, therefore, the cell harvest is greater. The indicated temperature range serves to completely disassociate the tissues of table 1, using the ratio of tissue mass / volume of the appropriate incubation medium and the protease concentrations indicated in the same table for each tissue. In no case can the temperature exceed 36 ° C, since at 37 ° C the protease-SF causes a fulminating digestion of the tissue. Below 32 ° C the dissociation is slower and it is necessary to lengthen the incubation time above 75 minutes, thereby increasing the cellular damage and obtaining a lower density of viable cells.

The incubation time of the tissue in the protease solution varies between 30 and 75 minutes, in inverse relation to the incubation temperature and the intensity of the mechanical aid, and in direct relation to the desired cell density. 4 ^a : Inhibition of enzymatic digestion

Protease-SF can be inhibited in two ways, depending on the post use of the cells; either by lowering the temperature of the cell suspension below 28 ° C, or by adding protease inhibitors to the cell suspension. Trypsin inhibitor obtained from soybeans, at 7μg / ml, or 10% fetal serum, are suitable inhibitors. The first form of inhibition is useful and cheap, when the cells are going to be spread on slides and fixed to be subjected to various techniques of cellular and molecular biology, such as autoradiography, immunocytochemistry, or in situ hybridization. The second way is indicated if the cells are intended to be cultured, or used for transplants. Analysis of results

The cell suspension resulting from the dissociation of the CNS tissues with the SF-protease, by the procedure described above, contains a majority of living cells of different shapes and a minority of dead cells, these being the inevitable result of the chopping processes of tissue and mechanical support. The analysis of the results includes a qualitative and quantitative evaluation of the cells obtained, and is performed by observation in the optical microscope using phase contrast

Qualitative analysis, consisting of the identification of cell types by their forms, reveals a great variety of cell types of both glial cells and neurons. Table 1 gives the qualitative analysis of the cell suspension resulting from the disintegration of the tissues indicated therein

The analysis of the number of viable cells in different ways, by the classical method of exclusion of tπpane blue indicates 85-96% of cells of the cell suspension that exclude the dye, that is, alive This analysis is performed during the last hour after inhibition of enzymatic digestion and without changing the cells of the medium, that is, in the same medium in which the dissociation has occurred This range of viability corresponds to the best results obtained in the dissociation of any of the types of tissue indicated in table 1 The percentage of live and morphologically identifiable cells varies depending on the type of tissue and animal species For example, in the disintegrated retinas of chicken embryonapses of 14-18 days of incubation 45-60% is obtained, while in the disaggregated retinas of the P5-P12 rat (stages of differentiation of the rat retina approximately equivalent to those indicated in the retinal po llo), a much lower percentage of 15-30% is obtained

A sene of experiences made with dissociated cells of different tissues with their morphologically preserved forms, both embutonated and adult, demonstrate that their cytoskeleton remains organized after the disintegration, which explains the preservation of their native forms. The experiences that demonstrate the preservation of the cytoskeleton consist in centigrade the cells on coverslips at 3000 rpm and fix them with 4% paraformaldehyde (PFA) in 0.1M sodium phosphate buffer, pH 7.2, to immunoreact them with monoclonal antibody 3CB2, marker of a protein associated with intermediate filament of the αthoskeleton, or fix them with 3% PFA plus 0.2% glutaraldehyde, 20 M EGTA and 0 065% Triton X-100 in sodium phosphate buffered saline, to immonurize them with anti-α-tubulin, or anti-β-actma. Subsequent immunoreaction of the cells with secondary antibodies bound to fluorescent molecules and observation in an optical fluorescence microscope, the three main molecule types (actma, tubuiin and intermediate filaments) are visualized, constituting the cellular α-skeleton. Since cells that dissociate with their native forms and exclude trypan blue, have the preserved plasma membrane and the organized cytoskeleton, the dissociation of cells with such a degree of integrity is only explained if SF-protease selectively breaks specific proteins of the extracellular matrix. This mechanism of action of the protease only takes place in a medium with very low ionic strength and at a temperature below 37 ° C as evidenced by experiments in which either the temperature or the ionic strength was modified

Advantages of the procedure object of this patent with respect to other procedures used to dissociate tissues from the CNS.

The dissociation of any of the tissues of the chicken or rat embryonic CNS, with the procedure object of the patent, provides live cells with the preserved cytoskeleton and the variety of forms that characterize them, while with any of the known procedures they obtain live cells, all of them round and not morphologically dentifiable.

With the procedure object of this patent, integral glial cells and neurons with their dendrites and long axon paths are obtained, which are inevitably cut during the tissue cutting, the cells in the process of differentiation even present their transient extensions and cones of increase. With the other known procedures the cells of the embryonic tissues are damaged in such a way that they only retain their somas, which explains why they are all round. The variety of cell types and the high number of whole cells that are obtained from the adult retina of the chicken and the rat, by dissociation with the process object of the patent, cannot be obtained by any of the other known procedures, being outside I argue that the SF-protease dissociates that part of the adult CNS better than any other protease. It is very likely that by making modifications to the procedure object of this patent, other tissues of the adult CNS can be dissociated with the SF-protease in the same way as the retina

The degree of integrity with which the embryonic cells are dissociated by the process object of this patent opens multiple ways of using them. The application of electrophysiology, autoradiography and immunocytochemical techniques, among others, to morphologically preserved dissociated cells is of immediate utility that adds precision to the analysis of the results, and opens up a range of possibilities in the design of experimentation aimed at obtaining basic knowledge in neurobiology development.

The high number of whole cells of different types, both embryonic and adult, that are obtained by the procedure object of this patent, opens the possibility of purifying any of the dissociated cell types for biochemical studies, which has been a permanent challenge in Neurobiology The use of embryonic cells with such degree of integrity for transplants of cells of the central nervous system, is advantageous with respect to the current use of mutilated cells obtained by disintegration with the other proteases and procedures. It is clear that morphologically preserved embryonic cells are more suitable for integration and development in a recipient tissue than damaged cells.

The use of whole embryonic cells to establish cell cultures that can more accurately simulate their functioning in situ is obviously more advantageous, and opens up new possibilities in the field of Biotechnology.

DESCRIPTION OF THE CONTENT OF THE DRAWINGS

To facilitate the understanding of the invention and as an integral part of this descriptive memo, the following figures are attached, which illustrate the following:

All drawings in Figures 1-4 have been made using a lucid camera coupled to a Zeiss optical microscope ("Universal" model).

Figure 1. Drawings of chicken embryo retinal cells from stages E17-

E18, dissociated by the protocol described in Example 1. The cells were spread on gelatinized slides, fixed with a mixture of ethanol, formalin and acetic acid (18: 1: 1), observed by phase contrast and drawn, f , photoreceptors in different phases of differentiation; h, horizontal cells; bi, bipolar cells; to cells amacrines of different types; g, ganglion cells; M, Müller cells (glia) in the process of differentiation. Magnifications: 800x.

Figure. 2. Drawings of adult rat retinal cells 1.5 to 18 months dissociated by the protocol described in example 2. The drawings were taken from fresh preparations, observed by phase contrast in the next two hours after of finishing the enzymatic digestion. c, cones (photoreceptors); b, canes (photoireceptors); bi, bipolar cells; a, amacrine cells; M, Müller cells (glia). Magnifications: 800x.

Figure. 3. Drawings of rat hippocampus cells of postnatal day 5 dissociated by the protocol described in example 3. The drawings were taken from fresh preparations, observed by phase contrast in the next two hours after the end of enzymatic digestion. . p, cells clearly identified as pyramidal. The rest of the unlabeled cells may belong to the pyramidal cell layer or to other layers of the hippocampus. Magnifications: 500x Fig. 4. Drawings of chicken embryo cerebellum cells of stage E14, dissociated by the protocol described in Example 4. The drawings were taken from fresh preparations, observed by phase contrast in the two hours following after finishing the enzymatic digestion. P, Purkinje cells in differentiation, in the phase in which the cell has short peπsomatic extensions. The rest of unlabeled cells may belong to the Purkinje cell layer or to other layers of the cerebellum. Magnifications: 500x.

EMBODIMENT OF THE INVENTION

For clarification, the following practical examples of carrying out the process object of the invention are given:

EXAMPLE 1

DISSOLVING THE CHICKEN EMBRYO RETINA CELLS OF THE STADIUM E17-E18

Dissociation medium:

Sucrose 175mM in 1mM sodium phosphate buffer at pH 6-6.2. This solution can be kept refrigerated openings days, at 4 ⁰ C.

Protease-SF solution:

1 mg of protease-SF of 24,000 UAP / mg dissolved in 2.4 ml of the dissociation medium. This solution is prepared daily and can be kept at room temperature until it is used, but not more than 3-4 hours.

Process:

A retinal (only the visual retina) is extracted from a 17 or 18 day incubation chicken embryo and placed in a glass well, with a capacity of 1 ml, containing 200 μl of dissociation medium.

With small scissors, with a fine and curved tip, the retina is chopped as finely as possible in the same well. With a micropipette of 1 ml, the contents of the well are passed to a glass tube containing. 100μ I of the protease solution and 300 µl of the dissociation medium, at a temperature of 34-35 ° C. Therefore, the tissue mass / volume ratio of the incubation medium is one retina in 600 μl. The protease concentration is 68 μg / ml.

The contents of the tube are carefully passed through the tip of a 1 ml micropipette 10 times in order to mechanically facilitate enzymatic dissociation. The tube is then placed in a 35 ° C water bath for 45 minutes, helping mechanically every 10 minutes

After the enzymatic digestion time, the bath tube is removed and left at room temperature (20-22 ° C), which inhibits the action of the SF-protease and the cells remain alive in the same medium in the that the incubation has been carried out for several hours (up to 4 hours).

Results:

The qualitative analysis of the results is performed by phase contrast observation in an optical microscope of transmitted light illumination. To do this, 20 µl of the cell suspension is taken, placed on a slide and a 24mm x 24mm coverslip is placed on top. The edges of the coverslip are sealed with nail lacquer, or liquid wax, to prevent the loss of liquid by evaporation and prevent the cells from leaving it. The cell suspension obtained by the described protocol contains a mixture of cells of all types in varying degrees of differentiation, with their native forms preserved, that is, photoreceptors, horizontal, bipolar, amacrine and ganglionic. The cells that appear intact, bright and with a refractory halo around their somas (45-60% of the dissociated) exclude the blue of the gut, which indicates that they are alive. In the cell suspension there are also, inevitably, round cells and cells that being recognizable by their forms have lost some of their extensions, product of the initial cutting of the tissue and of the mechanical help. 85-96% of these cells also exclude tπpane blue during the first hour after the end of enzymatic digestion. By way of illustration, drawings (Fig. 1) of a sample of intact cells are attached, representative of the multitude observed in the cell suspensions obtained by this protocol

EXAMPLE 2

Dissociation of cells from the adult rat retinal from 1.5 to 18 MONTHS

Dissociation medium: Sucrose 175 mM in double distilled water

This solution can be kept empty in a refrigerator for 4 days.

Protease-SF solution:

1 mg of protease-SF of 24,000 UAP / mg dissolved in 2.4 ml of the dissociation medium. This solution is prepared daily and can be kept at room temperature until it is used but no more than 3-4 hours.

Process:

A rat retina (only the visual retina) of 1.5 to 18 months of age is removed and placed in a glass well, with a capacity of 1 ml, containing 200 μl of dissociation medium.

With scissors of small size, with a fine and curved tip, the retina is chopped as finely as possible in the same well With a micropipette of 1 ml, the content of the well to a glass tube containing: 20 μl of the protease solution and 180 μl of the dissociation medium, at a temperature of 33 ° C. Therefore, the tissue mass / volume ratio of the incubation medium is one retina in 400 μl. The protease concentration is 21μg / ml. The contents of the tube are carefully passed through the tip of a 1 ml micropipette 10 times in order to mechanically facilitate enzymatic dissociation. The tube is then placed in a water bath at 33 ° C for 60 minutes, helping mechanically every 10 minutes.

After the enzymatic digestion time, the tube is taken out of the bath and left at room temperature (20-22 ° C), whereby the protease is inhibited and the cells remain alive in the same medium in which the incubation for several hours (up to 4 hours).

Results: The observation under the optical microscope, by phase contrast, of the cell suspension, shows abundant bright, refractory cells, with their preserved native forms. The tπpano blue test indicates that they are living cells. Among the morphologically preserved cells, numerous photoreceptors (cones and rods), bipolar cells, amacrines and Müller cells are identified. In the cell suspension there are also round cells and cells that, although identifiable due to their overall shape, have lost prolongations, as a result of the initial cleavage of the tissue and mechanical help during the enzymatic digestion process. In the figure. 2 drawings of a representative sample of the numerous morphologically preserved cells that are observed in the cell suspension obtained by the protocol of this example are presented.

EXAMPLE 3

DISCONNECTION OF POSTNATAL DAY RAT HIPOCAMPO CELLS 5

Dissociation medium:

Sucrose 300mM in double distilled water

This solution can be kept empty in a refrigerator for 4 days. Protease-SF solution:

Process:

The two hippocampus are extracted from a 5-day postnatal rat and placed in a glass well, with a capacity of 1 ml, which contains 300μ I of dissociation medium.

In the same well, slices of the hippocampus are made, as thin as possible, cutting them transversely with a scalpel, after which the slices are also chopped as finely as possible with small scissors, with a fine and curved tip.

With a micropipette of 1 ml, the well content is passed to a glass tube containing: 20 μl of the protease-SF solution and 480 μl of the dissociation medium, at a temperature of 33 ° C, at this time the digestion begins enzymatic Therefore, the tissue mass / volume ratio of the incubation medium is two hippocampus in 800 μl. The protease concentration is 10 μg / ml

The contents of the tube are carefully passed through the tip of a 1 ml micropipette 10 times, in order to mechanically help enzymatic dissociation. The tube is then placed in a 33 ° water bath for 35 minutes, helping mechanically every 5 minutes.

After the enzymatic digestion time, the bath tube is taken out and left at room temperature (20-22 ° C), whereby the protease is inhibited. The cells remain alive in the same medium in which the incubation has been carried out for 2 hours.

Results:

The optical microscope observation, by phase contrast, of the cell suspension shows abundant bright, refractory cells, with their native forms preserved. The trypan blue test indicates that these cells are viable. Among the morphologically preserved cells numerous pyramidal layer cells are identified in varying degrees of differentiation, and supposedly undifferentiated cells of this and other layers of the hippocampus. Round cells and cells are also found in the cell suspension, which, although they are identifiable due to their overall shape, have lost extensions, due to the initial cutting of the tissue and mechanical support during the incubation process. In Fig. 3, drawings of a representative sample of the morphologically preserved cells that are observed in the cell suspension obtained by the protocol of this example are presented.

EXAMPLE 4

DISSOLVING THE E14 STADIUM CHICKEN EMBRYON CELLS CELLS

Dissociation medium:

Sucrose 175mM in double distilled water This solution can be kept in vain for days in a refrigerator, at 4 ° C.

Protease-SF solution:

Process:

The entire cerebellum is removed from a chicken embryo for 14 days of incubation. It is placed in a glass well, with a capacity for 1 ml, which contains 300μ I of dissociation medium.

In the same well, slices of the cerebellum are made in the sagittal plane, as thin as possible, with a scalpel. The slices are chopped, also as finely as possible, with small scissors, with a fine and curved tip.

With a micropipette of 1 ml the content of the well is passed to a glass tube containing: 400 μl of the protease-SF solution and 500 μl of the dissociation medium, at a temperature of 34-35 ° C, starting at this time Enzymatic digestion Therefore, the tissue mass / volume ratio of the incubation medium is of a cerebellum in

1200 μl. The protease concentration is 136 μg / ml With a micropipette of 1 ml, it is helped mechanically 10 times, sucking and releasing the contents of the tube. The tube is then placed in a water bath, at a temperature of 34-35 ° C, for 30-45 minutes, helping mechanically every 10 minutes.

After the enzymatic digestion time, the bath tube is removed and left at room temperature (20-22 ° C), whereby the protease is inhibited. The cells remain alive in the same medium in which the incubation has been carried out for several hours (up to 4 hours)

Results:

The optical microscope observation, by phase contrast, of the cell suspension, shows abundant bright, refractory cells, with their native forms preserved. The tπpano blue test indicates that these cells are viable. Among the morphologically preserved cells numerous cells of the Purkinje layer are identified in varying degrees of differentiation, and supposedly undifferentiated cells of this and other layers of the cerebellar cortex. Round cells are also found in the cell suspension and cells that, being identifiable by their overall shape, have lost extensions, due to the initial cutting of the tissue and mechanical support during the incubation period. Figure 4 shows drawings of a representative sample of the morphologically preserved cells that are observed in the cell suspension obtained by the protocol of this example.

Claims

1.- Enzymatic procedure of dissociation of animal tissue cells, characterized by using an alkaline protease complex, designated "protease-SF", obtained by fermentation with a strain of Streptomyces fradiae, preferably Streptomyces fradiae ATCC 14544 or Streptomyces fradiae 10745

2.-Enzymatic method of dissociation of cells from animal tissues, according to claim 1 characterized in that it separates the cells, especially in CNS tissues of both embryonic and adult vertebrates, preserving their viability and their native structure and morphology.

3. Enzymatic method of dissociation of animal tissue cells, according to previous claims, characterized in that the fermentation takes place in an aqueous medium containing, in g / l, 30.0 of starch, 10.0 of soybean meal, 5 , 0 of "com steep solids", 1.0 of dipotassium phosphate, 0.25 of magnesium sulfate, 0.01 of ferrous sulfate and 10.0 of calcium carbonate; being the aerobic fermentation, in culture submerged with stirring, at 28-35 ° C and with a duration of 24-72 h

4. Enzymatic procedure of dissociation of cells of animal tissues, according to the preceding claims, characterized in that the proteases of the fermented broth are concentrated and purified by filtration or centrifugation thereof, precipitation with ammonium sulfate, chromatography and precipitation in aqueous solutions of acetone, thus obtaining a solid preparation of protease-SF

5. Enzymatic method of dissociation of animal tissue cells, according to the preceding claims, characterized in that the solid protease preparation contains the following characteristics a) A specific activity of 20,000-25,000 UAP / mg, the unit of proteolytic activity being defined (UAP) as the amount of protease-SF that, acting on denatured hemoglobin, releases, at pH 7.5, 25 ° C and in 10 min, hydrolysis products with a total absorbance, at 280 nm, equal to that of 1 μg of tyrosine b) Hydrolytic activity on many animal and vegetable proteins (hemoglobin, casein, gelatin, collagen, elastin, keratin, gluten, gliadin, etc.) c) Proteolytic activity at pH 5-12. d) Proteolytic activity not stimulated by compounds with sulfydryl groups or inhibited by compounds that block said groups, but slightly inhibited by trypsin inhibitors from soy and pancreas, and strongly inhibited by very low concentrations of isopropylfluorophosphate. e) Absence of contamination by glycosidases, amylases, lipases and nucleases. f) Great stability in solid state at room temperature. The stability in solution, although much lower than in the solid state, increases considerably in the presence of calcium ions and, under these conditions, is maximum at pH 8.2

6. Enzymatic method of dissociation of animal tissue cells, according to the preceding claims, characterized in that four essential stages are performed to disintegrate the CNS tissues of the different animal species:

I) .- Manual slicing of the tissue in a volume of dissociation medium containing 175-300 mM sucrose in 1 mM sodium phosphate buffer or in 5 mM HEPES buffer, at pH 6.2-7.4, or simply in distilled water at pH 6.4-6.5, and whose osmolality varies from 175 to 300 mOsm / Kg, at a temperature of 20-23 ^c C. II) .- Adding the suspension of the chopped tissue to the incubation medium, consisting in a solution of 4-156 μg / ml of protease-SF in the dissociation medium that is at a temperature of 28-36 ° C

III) .- Incubation of the tissue suspension in a water bath at a constant temperature between 32 ° C and 36 ° C for 30-75 min, mechanically facilitating tissue disintegration.

IV) .- Inhibition of enzymatic disintegration a) by lowering the temperature of the incubation mixture below 28 ° C, if the cells are to be spread on slides and fixed for use in cell or molecular biology techniques. b) Adding protease inhibitors such as trypsin inhibitor from soybeans, at 7 μg / ml, or 10% fetal serum, if the cells are destined for cultures or transplants. 7 - Enzymatic method of dissociation of cells from animal tissues, according to the preceding claims, characterized in that the precise values of osmolality and ionic strength of the dissociation and incubation means, tissue mass / volume ratio of the incubation medium, concentration of protease-SF in the incubation medium, temperature and pH of the incubation medium and duration of the incubation, are fixed depending on the type of tissue, animal species and stage of development or age of the animal

8 - Enzymatic method of dissociation of cells from animal tissues, according to previous claims, characterized in that it serves to disintegrate the embutonated retina of chicken and rat

9- Enzymatic method of dissociation of animal tissue cells according to claims pπmera to seventh, to disintegrate the adult retina of the chicken and the rat

10 - Enzymatic method of dissociation of animal tissue cells according to claims pnmera to seventh to disintegrate the hippocampal embπonaπo of the rat

11-Enzymatic procedure of dissociation of cells of animal tissues according to claims pπmera to seventh, to disintegrate the embryonic cerebellum of chicken and rat

12 - Use of dissociated cells of the embryonic CNS obtained by the enzymatic method of dissociation of animal tissue cells, according to claims pπmera to eighth, tenth and eleventh, characterized in that it is applied in a) cultures of neurons as models for development analysis, or pharmacological studies, b) neuronal transplants, c) the fine localization of molecules in subcellular structures, by cell and molecular biology techniques

13 - Use of dissociated cells of the adult CNS obtained by the enzymatic method of dissociation of animal tissue cells according to claims 7 to 9 and 9, characterized in that it is applied in a) the fine localization of molecules in subcellular structures by cellular and molecular biology techniques. b) electrophysiological studies

14. Use of dissociated cells of the embryonic and adult CNS obtained by the enzymatic method of dissociation of animal tissue cells, according to claims one to eleventh, characterized in that it is applied for the purification of specific populations of neurons or their precursor cells in biochemical studies and molecular genetics.

MODIFIED CLAIMS

[received by the International Bureau on December 13, 1997 (13.12.97), modified claims 1, 2, 4-6 and 12-14; other claims do not change (4 pages)]

1 - Eπzimatic procedure of dissociation of animal tissue cells, characterized by using a complete alkaline protease, designated "SF-protease". obtained by fermentation with a strain of Streptomyces fradiae, preferably Streptomyces fradiae ATCC 14544 or Streptomyces fradiae 10745

2-Enzymatic procedure of dissociation of cells of animal tissues, according to claim 1 characterized in that it separates the cells, especially those of the CNS tissues of both embryonated and adult vertebrates, preserving their viability and their native structure and morphology

3 - Enzymatic method of dissociation of animal tissue cells, according to previous claims, characterized in that the fermentation takes place in an aqueous medium containing, in g / l, 30.0 of starch, 10.0 of soybean haπna, 5, 0 of "com steep solids", 1.0 of dipotassium phosphate, 0.25 of magnesium sulfate, 0.01 of ferrous sulfate and 10.0 of calcium carbonate, being aerobic fermentation, in submerged culture with stirring, at 28- 35 ° C and with a duration of 24-72 h

4-Enzymatic procedure of dissociation of animal tissue cells, according to the preceding claims, characterized in that the proteases of the fermented broth are concentrated and puffered by filtration or centπfugaαon thereof, precipitation with ammonium sulfate, chromatography and precipitation in aqueous solutions of acetone , thus obtaining a solid preparation containing the protease complex called protease-SF

5 Enzymatic method of dissociation of animal tissue cells, according to the preceding claims, characterized in that the solid protease preparation contains the following characteristics a) A specific activity of 20000-25,000 UAP / mg, defining the unit of proteolytic activity (UAP) ) as the amount of protease-SF that, acting on denatured hemoglobin, releases, at pH 7.5, 25 ^β C and in 10 mm, hydrolysis products with a total absorbency, at 280 nm, equal to that of 1 μg of tyrosine b) Hydroli ca activity on many animal and vegetable proteins (hemoglobin, casein, gelatin, collagen, elastin, keratin, gluten, gliadin, etc.) 2k

c) Proteolytic activity at pH 5-12 d) Proteolytic activity not stimulated by compounds with sulfidyl groups or inhibited by compounds that block said groups, but slightly inhibited by tπpsma inhibitors from soybeans and pancreas, and strongly inhibited by very low concentrations of isopropylfluorophosphate e) Absence of contamination by g cosidases, amylases, lipases and nucleases f) High stability in solid state at room temperature Solution stability, although much lower than in solid state, increases considerably in the presence of calcium ions and under these conditions it is maximum at pH 8.2

6 - Enzymatic method of dissociation of cells from animal tissues, according to the preceding claims, characterized in that four essential stages are performed to disintegrate the CNS tissues of the different vertebrate animal species I) - Manual cutting of the tissue in a volume of dissociation medium containing 175-300 mM sucrose in 1 mM sodium phosphate buffer or 5 mM HEPES buffer, at pH 6.2-7.4, or simply in distilled water at pH 6.4-6.5, and whose osmola vadad of 175 to 300 mOsm / Kg, at a temperature of 20-23 ^o C II) - Addition of the suspension of the chopped tissue to the incubation medium, consisting of a solution of 4-156 μg / ml of protease-SF in the dissociation medium that is at a temperature of 28-36 ° C

III) - Incubation of the tissue suspension in a water bath at a constant temperature between 32 ° C and 36 ° C for 30-75 min, mechanically facilitating tissue disintegration IV) - Inhibition of enzymatic disintegration a) Lowering the incubation mixture temperature below 28 ° C, if cells are to be spread on slides and fixed for use in cell or molecular biology techniques b) Adding protease inhibitors such as tπpsin inhibitor from soybeans , at 7 μg / ml, or 10% fetal serum, if the cells are destined for cultures or transplants

7 - Enzymatic method of dissociation of animal tissue cells, according to the preceding claims, characterized in that the precise values of osmolality and ionic strength of the dissociation and incubation media, tissue mass / volume ratio of the incubation medium, protease-SF concentration in the incubation medium, temperature and pH of the incubation medium and incubation duration, are set at dependence on the type of tissue, animal species and stage of development or age of the animal.

8. Enzymatic method of dissociation of animal tissue cells, according to previous claims, characterized in that it serves to disintegrate the embryonic retina of the chicken and the rat.

9- Enzymatic method of dissociating cells from animal tissues, according to claims one to seven, to disintegrate the adult retina of the chicken and the rat.

10 - Enzymatic method of dissociation of animal tissue cells, according to claims one to seven, to disintegrate the embryonic hippocampus of the rat.

11. Enzymatic method of dissociation of animal tissue cells, according to claims one to seven, to disintegrate the embryonic cerebellum of chicken and rat.

12.-Enzymatic procedure for obtaining cells of embryonic tissues of the CNS of animals, according to claims first to eighth, tenth and eleventh, which are used in the following applications: a) cultures of neurons as models for development analysis embryonic, or pharmacological studies, b) neuronal transpiants, c) the fine localization of molecules in subcellular structures, by cellular and molecular biology techniques.

13 - Enzymatic procedure for obtaining adult tissue cells from the CNS of the animals, according to first and seventh and ninth claims, which are used in the following applications. a) the fine localization of molecules in subcellular structures by cellular and molecular biology techniques, b) electrophysiological studies.

14 - Enzymatic procedure of dissociation of cells of embryonic and adult tissues of the CNS of animals, according to claims one to eleventh, for the purification of specific populations of neurons, or of their precursor cells in biochemical and molecular genetic studies.