EP1084229A1 - Herstellung von primmorphen aus dissoziierten zellen von schwämmen, korallen und weiteren invertebraten und deren verwendung - Google Patents

Herstellung von primmorphen aus dissoziierten zellen von schwämmen, korallen und weiteren invertebraten und deren verwendung

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Publication number
EP1084229A1
EP1084229A1 EP99955288A EP99955288A EP1084229A1 EP 1084229 A1 EP1084229 A1 EP 1084229A1 EP 99955288 A EP99955288 A EP 99955288A EP 99955288 A EP99955288 A EP 99955288A EP 1084229 A1 EP1084229 A1 EP 1084229A1
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Prior art keywords
primmorphs
cells
aggregates
cell
substances
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EP99955288A
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German (de)
English (en)
French (fr)
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Werner E. G. MÜLLER
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • This invention relates to the establishment of the first and thus a new method for the cultivation of sponge cells, coral cells and cells of other invertebrates in vitro.
  • the cells cultured in vitro which can be cultured as aggregate-like units, are called primmorphs.
  • This is a method that makes it possible for the first time to introduce processes with cells / aggregates / primmorphs from sponges, corals and other invertebrates: (i) for the production of proliferation and DNA synthesis modulating substances, (ii) for identification / Detection of environmentally harmful substances, (iii) for the cultivation of bacteria and other microorganisms, (iv) for the production of asexual reproductive bodies, which can be used in aquaculture to grow out into corresponding organisms, (v) for the creation of cell banks, (vi) for the optimization of the Nutritional needs of the cells / aggregates / primmorphs, and (vii) to identify substances that modulate telomerase activity in cells / aggregates / primmorphs.
  • the Phylum Porifera (sponges) has a monophyletic origin with the other Metazoen-Phyla [Müller WEG (1995) Naturwissenschaften 82, 321-329].
  • a basic autapomorphic feature of the Metazoa, including the Porifera, is, for example, the presence of the receptor tyrosine kinase, which can only be found in Metazoa [Müller WEG, Schcke H (1996) Prog Molec Subcell Biol 17, 183-208].
  • the Porifera show a plesiomorphic feature that cannot be found in any other higher Metazoan phylum; all (or almost all) of their cells have high levels of telomerase activity [Koziol C, Borojevic R, Steffen R, Müller WEG (1998) Mech Aging Develop 100, 107-120]. In principle, this indicates that sponge cells cannot be divided into germ or somatic cells [Müller WEG (1998a) Progr Molec Subcell Biol 19, 98-132; Müller WEG (1998b) Naturwiss 85: 11-25]. In higher non-cancer patients Metazoa are almost always the germ cells telomerase-positive, while the somatic cells are telomerase-negative [Lange T v (1998) Science 279, 334-335].
  • Dendronephthya hemprichi (Cnidaria, Anthozoa, Alcyonaria) acquired from a pet store and - with the exception of temperature (22 ° C) - kept like sponges.
  • the remaining pieces of tissue are again mixed with new CMFSW-E in the same ratio and again shaken gently rotating.
  • the supernatant is decanted and discarded.
  • the tissue pieces are now mixed with new CMFSW-E and moved on a shaker for usually 40 minutes.
  • the supernatant, which contains the cells, is through a nylon mesh, mesh size z. B. 40 microns, filtered and collected in a tube. This operation, shaking the tissue Pieces in CMFSW-E and filtration of the supernatant through a nylon mesh is repeated several times.
  • the combined cell suspensions are gently centrifuged to obtain the cells [usually at 500xg for 5 min].
  • the Zeil suspension is taken up in seawater / antibiotic solution [antibiotics: usually 100 IU penicillin and 100 ⁇ g / ml streptomycin]. This process is usually repeated one or more times.
  • the collected cells are obtained by centrifugation. A cell suspension of 10? Cells / ml is adjusted and 1 ml thereof is usually added to 5 ml sea water / antibiotic solution in a z. B. 60 mm petri dish [e.g. B. Falcon catalog number 3004].
  • the cells are incubated, usually at 16 ° C, and are usually transferred four times to a new Petri dish by careful resuspension with seawater / antibiotic solution. This is also to ensure that the cells do not stick to the shell.
  • New aggregates can be obtained from the mother petri dish several times, usually two to five times.
  • the washed aggregates remain in the Petri dishes until they have a smooth surface.
  • the seawater / antibiotic solution is usually renewed every day; two thirds of the solution are replaced by fresh sea water / antibiotic solution.
  • the incorporation of BrdU [5-bromo-2'-deoxy-uridine] into the cellular DNA was measured using the "BrdU labeling and detection kit", according to the manufacturer's instructions.
  • the primmorphs are usually in 1 ml
  • the cells are then incubated with anti-BrdU mouse monoclonal antibodies and the immune complex is made visible with anti-mouse Ig / coupled with alkaline phosphatase and with the color substrate nitro blue tetrazolium salt.
  • the cells are analyzed by light microscopy.
  • the primmorphs are fixed in 4% paraformaldehyde / phosphate-buffered saline [Romeis, B. (1989) Microscopic Technology. Kunststoff; Urban and Schwarzenberg]. After dehydration with ethanol, the primmorphs are embedded in Technovit 8100 [Beckstead J H (1985) J Histochem Cytochem 9, 954-958]. Sections with a thickness of 2 ⁇ m are made and stained with Ziehl's Fuchsin solution [Martoja R, Martoja M (1967) Initiation aux Techniques de THistologie Animale. Prem. Ed. Masson et Cie, Paris].
  • Telomerase activity is detected using the polymerase chain reaction [PCR] method using the "Telomerase Detection Kit (TRAPeze)"; Details have been described earlier [Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Science 266: 2011-2014; Koziol C, Borojevic R, Steffen R, Müller WEG (1998) Mech Aging Develop 100, 107-120]. The cell extracts added correspond to 5 x 10 3 cell equivalents.
  • the amplification products are quantified by electrophoresis using a 12.5% non-denaturing polyacrylamide gel in 0.5 x TBE buffer [performed according to the manufacturer's instructions].
  • the gels are stained with SYBR Green I in order to recognize DNA fragments [Molecular Probes (1996) MP 7567 07/16/96].
  • the signals are recorded with a GS-525 Molecular Imager (bio Wheel) quantified.
  • the degree of telomerase activity is given in TPG (total product generated) and is calculated as described [Oncor (1996) TRAPeze telomerase detection kit; catalog no. S7700 kit; second edition. Oncor, Gaithersburg, MD; UNITED STATES].
  • Specimens of the S. domuncula sea sponge were used to isolate the cells (Fig. 1A). Single cells were obtained by dissociation as described above. After the specified washing steps, the protozoa that preferentially adhere to the surface of the plastic culture vessels are removed. The cells are transferred to a seawater / antibiotic solution. After the entire treatment / incubation period of usually five days, primmorphs (Fig. 1 D) form from the cell aggregates (Fig. 1 B and C). After an incubation period of two days, the diameter of the cell aggregates is approximately 100 ⁇ m (Fig. 1B) and increases in size continuously; after four days a diameter of usually 300 ⁇ m is reached (Fig. 1 C). During this time, the aggregates are rounded off. Usually, primmorphs about 1 to 2 mm in size form after another three to five days (Fig. 1 D).
  • the cells that form the squamous epithelium of the primmorphs are pinacocytes, as can be deduced from their flattened fusiform runners and their clear nuclei [Review: Simpson TL (1984) The Cell Biology of Sponges. Springer Verlag, New York]; the size of the cells fluctuates around 20-30 ⁇ m.
  • the cells within the primmorphs are mainly spherical cells. They have a diameter of approximately 30 to 40 ⁇ m and are characterized by large round vacuoles, which occupy the majority of the cells.
  • the other cells can be called amoebocytes and archaeocytes and have a size of about 40 ⁇ m.
  • the outer appearance of the primmorphs is smooth and almost spherical (Fig. 1 D); the histological sections show that the cells in the primmorphs are well organized into a tissue-like body (Fig. 1 E and F). It is striking that the squamous epithelium is formed from a multicellular layer of mostly pinacocytes (Fig. 1 F), which cannot be found in natural sponges; these are limited by a unicellular epithelium.
  • the organized arrangement of the cells within the primmorphs also distinguishes them from the aggregates which are formed from dissociated cells in the presence of the homoigenic aggregation factor [Müller WEG (1982) Intern Rev Cytol 77, 129-181].
  • the primmorphs described here are formed from a single cell suspension in vitro. As shown here, the dissociated cells form tissue-like bodies.
  • the construction of functional primmorphs from a single cell suspension implies that this formation is an active process that involves the removal of dead cells and cell fragments; when viewed microscopically, this can be seen as "frayed" edges of the primmorphs.
  • the formation of a squamous epithelium from pinacocytes further indicates that sponge cells differentiate and then re-differentiate into cells that are necessary for the formation of primmorphs.
  • the primmorphs obtained from S. domuncula have been kept in culture for over five months. These primary primmorphs can be dissociated into single cells again in CMFSW-E. The resulting single cell suspension is still able to form aggregates and then primmorphs, which are now called secondary primmorphs. This process occurs when the cells are transferred to sea water / antibiotic solution.
  • the kinetics of primmorph formation is usually identical to that observed for primary primmorphs.
  • the cells from primary primmorphs adhere weakly to the surface of the culture vessels after dissociation [e.g. B. Falcon catalog number 3004].
  • the tubes must be roughened slightly with a cover glass or a rubber scraper.
  • the level of telomerase activity was determined in the cells during the formation of primmorphs from a single cell suspension.
  • the results show that cells in the natural cell structure have a high telomerase activity; a quantitative analysis showed an activity of 8.9 TPG units / 5 x 10 3 line equivalents (Fig. 2; lane a). If the telomerase activity was determined in cells which were left in the dissociated single-cell state for 14 hours, the enzyme level dropped to 0.9 TPG units / 5 ⁇ 10 3 cell equivalents (FIG. 2; lane b). However, when cells from primmorphs [about 10 days after formation from single cells] were used for the analysis, a telomerase activity of 4.7 TPG units / 5 ⁇ 10 3 cells resulted (FIG. 2; lane c).
  • the BrdU label and the "Detection" approach were used to show that those cells that had organized themselves into primmorphs regain their proliferation ability.
  • the cells were incubated with BrdU for 12 hours determined by BrdU in the DNA and quantified with the aid of an anti-BrdU monoclonal antibody as described above Cells which have incorporated BrdU in the DNA are distinguished by a dark (dark brown) colored cell nucleus (FIGS. 3B-D). Controls that were not incubated with the primary antibody against BrdU are not stained (Fig. 3A).
  • Fig. 4 The analysis of tissue samples after extraction of the RNA and subsequent separation in the agarose gel shows that in addition to the two host rRNAs, 28S and 18S rRNA, prokaryotic rRNAs, 23S and 16S rRNA also occur in S. domuncula (Fig. 4; Lane a). After dissociation and subsequent production of primmorphs in culture, the prokaryotic rRNAs, 23S and 16S rRNA, are usually also detected (Fig. 4; lane b). This shows that primmorphs can also contain microorganisms without "contaminating the culture".
  • the percentage of BrdU-positive cells as well as two-nucleus stages is given for each batch series.
  • the analysis was performed with: (i) dissociated cells kept in CMFSW-E for 1 day, (ii) cell aggregates from single cell suspension after 1 day in culture in sea water, (iii) primary primmorphs [formation after 10 days ] and (iv) secondary primmorphs [formed from one month old primary primmorphs after dissociation in single cells and subsequent formation of secondary primmorphs]. 300 cells were counted per batch.
  • the primary primmorphs were obtained from single cells of S. domuncula and used for the experiments after 21 days. 4.1. Identification of DNA synthesis modulating and proliferation modulating substances using primmorphs
  • the primmorphs were incubated for two days with different concentrations of PMA.
  • the DNA synthesis was then determined using BrdU labeling as described above.
  • the control percentage of BrdU positive cells in primmorphs without test substance, was set as 100%.
  • the results which are summarized in Fig. 5, show that in the concentration range from 0.01 to 1 ⁇ g PMA / ml there is a marked percentage increase in BrdU-positive cells in primmorphs (Fig. 5). Based on this data, it can be concluded that primmorphs are excellent
  • Organisms can be assumed that the system of the primmorphs is a sensitive one
  • the bioactive substances of primmorphs are produced in small (1 ml) or larger (20 l) quantities; it can also take place in larger bioreactors and also in aquaculture.
  • the cells that are apoptotically killed by heat shock (which serve as a source of nutrients) expose phosphatidylserine and phosphatidylinositol and other lipids on their cell surface. These apoptotically modified cells / membranes are phagocited by the primmorphs.
  • Tumor cells are known to have high telomerase activity [Hastie ND, Dempster M, Dunlop AG, Thompson AM, Green DK, Allshire RC (1990) Nature 346, 866-868].
  • One goal of medical research is to reduce the activity of this enzyme in tumor cells. Due to the fact that sponge cells are telomerase-positive, the model of the primmorphs is therefore extremely suitable for testing substances that reduce this activity.
  • telomerase activity can be modulated in cells of primmorphs.
  • telomerase activity in cells of S. domuncula after adding antibody (rabbit - polyclonal) to primmorphs. 50 ⁇ l of antibody per well were added to the batches. The telomerase activity is given in TPG (total product generated) and standardized to 5 x 10 3 cell equivalents.
  • Primmorphs can be used for the cultivation of larger tissue cultures and for the cultivation of entire corresponding organisms. Aquaculture is used for this. Primmorphs are usually incubated in z. B. "culture chamber slides" [(Nunc) - catalog number 177453] with these into larger artificial containers (such as aquariums), or directly into nature (aquatic environment). If larger artificial containers (such as aquariums) are used, as in the case of S. domuncula, these are filled with artificial sea water and mixed with the usual trace minerals. Usually once a week the medium is some organic material, such as. B. tuna added. After an average of one month, the primmorphs have grown to 5 mm large organism-like structures. Longer incubation times lead to further growth until the finished organism.
  • cells either immediately after dissociation from the parent organism, e.g. B. the sponge S. domuncula, or obtained from primmorphs and frozen.
  • the cells are slowly frozen (usually 1 ° C./min) in a suitable tube, usually in dimethyl sulfoxide (usually 10% in sea water) or glycerol (usually 20% in sea water).
  • the cell number is adjusted to approximately 5 x 10 6 cells / ml.
  • the cells are then transferred to liquid nitrogen and can be stored for over six months. If the cells are thawed again, they can be opened in stages
  • Invertebrates can be frozen. This also gives you the option of sending cells in the usual way (e.g. in dry ice). This means that cells can be cultivated at a central location, i.e. cell banks can be established.
  • FIGURES Fig. 1 Formation of primmorphs from cells of the Suberites domuncula sponge.
  • A. a specimen of S. domuncula; Magnification x 1.
  • the cells are dissociated by treatment in CMFSW-E. After two days in culture, cell aggregates [medium: seawater / antibiotics] B form, which increase in size [uptake after three to four days] C; x 10.
  • Primmorphs D usually form after five days; x 10.
  • E and F cross-sections through a primmorph, show the multi-cell epithelium-like location of pinacocytes that surrounds the inner part, which consists of spherical cells, amoebocytes and archaeocytes; E: x 20; F: x 45.
  • Fig. 2 Telomerase activity in cells of S. domuncula. Telomerase activity was determined (i) in cells of tissue (lane a), (ii) in single cell suspension [the cells were analyzed for 14 hours] (lane b) and (in) in primmorphs (lane c). Defined amounts of material, corresponding to 5 x 10 3 line equivalents, were created incubated in the TRAP approach. After PCR amplification, the products were separated in a non-denaturing polyacrylamide gel; the gel was stained with SYBR Green I to make the DNA fragment visible.
  • Fig. 3 Cells from primmorphs with the aim of detecting DNA synthesis there.
  • the primmorphs were incubated in BrdU (details in the text); BrdU is incorporated into the DNA - if DNA synthesis has taken place.
  • the built-in BrdU units are detected by an antibody reaction using the "BrdU labeling and detection kit". These appear as dark spots that mark the core of the line.
  • B - D cells from primmorphs which were incubated with BrdU and then treated with the identification reagent to detect BrdU.
  • Fig. 3D shows a BrdU-positive cell, indicated by an arrow; a BrdU-negative cell is marked by an arrow head.
  • Fig. 4 Analysis of tissue samples and primmorphs on rRNA. The material was extracted and the RNA was then separated in the agarose gel. In addition to the two eucaryotic host rRNAs [euc], 28S and 16S rRNA, procaryotic rRNAs [proc], 23S and 16S rRNA are also visualized by ethidium bromide.
  • Fig. 5 Influence of the phorbol ester Phorbol 12-myristat 13-acetate (PMA) on the percentage of BrdU-positive cells in primmorphs of S. domuncula. The primmorphs were incubated with different concentrations of PMA for two days. The DNA synthesis was then determined using BrdU labeling. The control, percentage of BrdU-positive cells in primmorphs without test substance, was set to 100%. 300 cells were counted per batch.
  • PMA phorbol ester Phorbol 12-myristat 13-acetate
  • Fig. 6 Influence of cadmium, concentrations between 0.1 ng / ml and 100 ng / ml were selected, on the level of the percentage of BrdU-positive cells in primmorphs.
  • Fig. 7 Production of suberitine, a toxic protein, in primmorphs from S. domuncula. Primmorphs were cultivated. After 0 [transfer of the primmorphs into the 24-hole plates] to 20 days, primmorphs were removed and tested for bioactivity. For each incubation point five parallel batches of primmorphs were taken, a crude extract was prepared and tested for hemolytic activity. The titer, expressed in UU (hemolytic units), is based on 1 mg protein extract. The mean values with the standard deviations are given. * (P ⁇ 0.001).

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EP99955288A 1998-05-30 1999-05-06 Herstellung von primmorphen aus dissoziierten zellen von schwämmen, korallen und weiteren invertebraten und deren verwendung Withdrawn EP1084229A1 (de)

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DE19824384A DE19824384B4 (de) 1998-05-30 1998-05-30 Herstellung von Primmorphen aus dissoziierten Zellen von Schwämmen und Korallen, Verfahren zur Kultivierung von Zellen von Schwämmen und Korallen zur Produktion und Detektion von bioaktiven Substanzen, zur Detektion von Umweltgiften und zur Kultivierung dieser Tiere in Aquarien und im Freiland
DE19824384 1998-05-30
PCT/EP1999/003121 WO1999063060A1 (de) 1998-05-30 1999-05-06 Herstellung von primmorphen aus dissoziierten zellen von schwämmen, korallen und weiteren invertebraten und deren verwendung

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CA (1) CA2332968A1 (ja)
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ES2261054B1 (es) * 2004-12-31 2007-11-16 Universidad De Almeria Procedimiento para obtener un extracto de pulpo, el producto obtenido y su aplicacion como suplemento para el cultivo y criopreservacion de tejido y de celulas en suspension de invertebrados marinos.
US20110179504A1 (en) * 2007-09-17 2011-07-21 Ramot at Tel-Aviv Univeristy Ltd. Method For Coral Tissue Cultivation And Propagation
RU2472516C1 (ru) * 2011-06-16 2013-01-20 Общество с ограниченной ответственностью "АйБИОСТ" Биоматериал для замещения костных дефектов
CN102630646B (zh) * 2012-04-24 2013-07-17 海南大学 一种枝状珊瑚人工感染法
JP6404950B2 (ja) 2015-01-21 2018-10-17 ギル メディカル センターＧｉｌ Ｍｅｄｉｃａｌ Ｃｅｎｔｅｒ ホスファチジルイノシトールリン酸結合物質の細胞死滅検出用途
CN105076115A (zh) * 2015-08-31 2015-11-25 厦门大学 海绵细胞团冷冻保存液及其保存方法
CN106973889B (zh) * 2016-01-04 2018-08-24 南京三生生物技术股份有限公司 一种白血病治疗用的细胞冻存液
CN107027742B (zh) * 2016-01-05 2019-01-18 河北生命原点生物科技有限公司 一种用于白血病治疗的细胞冻存液
FR3109584B1 (fr) * 2020-04-27 2024-05-10 Paulou Massat Sophie De Microcompartiment pour la culture de cellules de cnidaires

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CA2332968A1 (en) 1999-12-09
DE19824384B4 (de) 2006-03-30
AU754250B2 (en) 2002-11-07
NO20006051L (no) 2001-01-30
WO1999063060A1 (de) 1999-12-09
NZ508602A (en) 2002-10-25
DE19824384A1 (de) 1999-12-02
AU4258599A (en) 1999-12-20
JP2002517188A (ja) 2002-06-18
NO20006051D0 (no) 2000-11-29

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