Immortalized cell

Abstract

The present invention relates to an immortalized cell, an immortalized cell line comprising said immortalized cell, a cell culture comprising the immortalized cell or cell line, and a method for the production of an immortalized cell.

Claims

1. An isolated, in vitro immortalized cranium periosteum cell, wherein the cell was deposited on 29 Nov. 2013 under the designation Tag58 at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany (DSMZ) under the accession number DSM ACC3218.

2. A cell culture, which comprises a plurality of the isolated, in vitro immortalized cranium periosteum cells of claim 1.

3. A method for the production of an isolated, in vitro immortalized cranium periosteum cell deposited on 29 Nov. 2013 under the designation Tag58 at the Deutsche Sammlunq von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany (DSMZ) under the accession number DSM ACC3218, comprising the following steps: 1) providing an isolated cranium periosteum primary cell, and 2) immortalizing the cranium periosteum cell by transduction of the large T-antigen of the SV40 virus into the cranium periosteum primary cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a Western blot for detection of the permanent immortalization state of the periosteum cell according to the invention (Tag58) via the detection of the large T-antigen of SV40. A: Primary cranium periosteum cells (M58) at 37 C.; B: TAg58 at 33 C.; C: TAg58 at 37 C.; D: TAg58 at 38 C.; E: TAg58 at 39 C.

(2) FIG. 2 shows the result of a quantitative PCR for the detection of the gene expression of the human telomerase reverse transcriptase (hTERT). Posco: positive control; Tag58 co: Tag58 undifferentiated; M58 co: M58 undifferentiated; TAg58 OB: Tag58 osteogenically stimulated (day 10); M58 OB: M58 osteogenic stimulated (day 10):

(3) FIG. 3 shows the result of a quantitative PCR for the detection of the gene expression of osteogenesis-relevant markers. A: alkaline phosphatase; B: osterix; C: runx-2; D: osteocalcin. The ratio of expression level of the respective gene to that of the housekeeping gene GAPDH on day 3, 10 and 20 of the osteogenesis is shown.

(4) FIG. 4 shows the result of a microscopic examination for the detection of calcium phosphate precipitates with the aid of alizarin staining at different examination time points of the osteogenesis (day 16, d 16; day 20, d20; day 24, d 24; day 30, d 30; day 37, d 37), CO: undifferentiated cells; OB: osteogenically stimulated cells.

(5) FIG. 5 shows the result of a microscopic examination with the aid of the osteolmage staining on day 37 (d 37) of the osteogenic stimulation for the detection of calcium phosphate precipitates. CO: undifferentiated cells; OB: osteogenetic stimulated cells.

(6) FIG. 6 shows the result of a flow cytometric examination for the detection of characteristic surface antigenes of the cell line in comparison to the parental cells.

DESCRIPTION OF PREFERRED EMBODIMENTS

1. Material and Methods

(7) Lentiviral Transduction of Primary Cranium Periosteum Cells

(8) Primary cranium bone skin cells or cranium periosteum cells (8 aliquots of 5.010.sup.8 cells) were sent on dry ice to the company Sirion Biotech, Martinsried, Germany, which lentivirally transduced them. This occured by using the vector Lenti_pCDH-CMV-LTtsA58-EF1-Neo replic.-defic., self-inactivating 3rd generation lentiviral vector.

(9) 72 hours after the transduction the selection of the transduced cells with 0.50 mg/ml G418 was started. Under this selection pressure the cells were cultivated for 10 days and then cryopreserved (in Cryo-SFM medium of Promocell, C-29912). Non-transduced cells died after 5 days of cultivation in the geneticin-(G418)-containing medium. After thawing the cells maintain the immortalization state at 37 C.

(10) The induction of the osteogenic differentiation was carried out by the addition of 4 M dexamethasone, 10 mM -glycerophosphate disodium salt hydrate and 100 M ascorbic acid (L-ascorbic acid 2-phosphate). The formation of calcium phosphate precipitates by the cell monolayer is already visible after 15 days.

(11) As already described above, the primary cells were isolated from the cranium bone skin. First, the tissue was mechanically comminuted, then the enzymatic digestion of the tissue occurred with type XI collagen for 90 min. The individual cells were cultured in 75 cm.sup.2 tissue culture bottles in DMEM:Ham's F12 (1:1 mixture)+10% FCS+1% PenStrep+fungicides.

(12) The expressed transgene is the cDNA of the large T-antigen (LTtsA58) of polyomavirus SV40. 72 hours after the transduction of the primary cranium periosteum cells the latter were selected for 10 days in the presence of the antibacterially active substance geneticin ((3-418; 0.5 mg/ml) and then cryopreserved.

(13) The cells were subjected to an immortalization protocol which should lead to a conditioned immortalization. Accordingly, it was expected that a cell cultivation at 33 C. is necessary for maintaining the immortalization state. At higher temperatures of about 37 C. to 39 C. the transgene should be deactivated and the cells should acquire their basic state again. However, surprisingly this could not be verified in the experiments of the inventor. The cells showed an immortalization state regardless of the cultivation temperature.

(14) In order to improve comparability of the results, both immortalized and primary cells were cultured and examined at 37 C.

(15) So generated immortalized cranium periosteum cells were frozen and sent to the Leibnitz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstrae 7B, 38124 Braunschweig, Germany. There, the viability and purity of the material was confirmed. The cell culture with the designation Tag58 was officially deposited at the DSMZ on 29 Nov. 2013 under the accession number DSM ACC3218.

2. Results

(16) Detection of the Immortalization State by Means of Western Blotting

(17) In order to prove that the generated Tag58 cells are actually immortalized, the large T-antigen of SV40 was detected at the protein level in the lysates of the cells by Western blotting.

(18) Due to the aim for conditioned immortalization it was assumed that the large T-antigen of SV40 can be detected only in cultured cells at 33 C. and not at other incubation temperatures, such as 37 C., 38 C. or 39 C. However, this could not be verified (see FIG. 1). The SV40 T-antigen was detectable independent of the incubation temperature in all samples of the immortalized cell line (lanes B to E). In the cell lysate of the parental primary cells no specific SV40 T-antigen signal was detectable (lane A).

(19) The expected size of the specific SV40 T-antigen band of about 80 kDa was verified. As an internal control the housekeeping protein GAPDH was used.

(20) Detection of the Gene Expression of Human Telomerase Reverse Transcriptase (hTERT) by Means of Quantitative PCR

(21) In order to test whether the SV40 large T-antigen integrated into the genome is actually functionally active, the gene expression of p53 and hTERT was examined by quantitative PCR, For this purpose the Tag58 cells according to the invention were osteogenically stimulated. The induction of osteogenic differentiation occured by the addition of 4 M dexamethason, 10 mM -glycerophosphate disodium salt hydrate and 100 M ascorbic acid pascorbic acid 2-phosphate). For the mRNA levels of p53 no significant differences were detected in the Tag58 cells in comparison to the parental M58 primary cells. In contrast to this, however, the hTERT gene expression could be detected only in the Tag58 cells (see FIG. 2). The specific hTERT band showed at day 10 an even stronger signal in the osteogenically stimulated Tag58 cells (Tag58 OB).

(22) Both in the undifferentiated (M58 Co) and the osteogenically stimulated parental M58 cells (M58 OB) no specific signal for hTERT was detected.

(23) Detection of the Gene Expression of Osteogenic Relevant Markers by Means of Quantitative PCR

(24) The gene expression of the early osteogenesis marker alkaline phosphatase and the transcription factors osterix and runx-2 and the late osteogenesis marker osteocalcin was quantitated by means of PCR. The expression of the osteogenic differentiation was examined at the beginning (day 3; T3) in the middle (day 10; T10) and at the end (day 20; T20).

(25) As shown in FIG. 3A, at the beginning and in the middle of osteogenesis (day 3 and 10) both higher basal level (Co) and higher inductions (OB) of alkaline phosphatase in the Tag58 cells were detectable by PCR. At the end of the differentiation (day 20) the mRNA levels become again more similar.

(26) At all examined time points of osteogenesis higher basal level (Co) of the transcription factor osterix could be detected in the immortalized cells in comparison to the primary cells (FIG. 3B). With the exception of day 10 higher inductions (OB) were also detected in the Tag58 cells.

(27) The quantitative examination of runx-2 gene expression showed at all three examined time points of osteogenesis higher levels in the Tag58 cells in comparison to the parental primary cells M58 (FIG. 3C).

(28) The analysis of osteocalcin gene expression showed only on day 10 and 20 higher basal levels in the Tag58 cells, as can be seen in FIG. 3D.

(29) Colorimetric Detection of Proliferation Activity

(30) The detection of the proliferation activity was made by means of a semi-quantitative MTT-based colorimetric assay (EZ4U, Biozol). Through the conversion of tetrazolium salt to formazan derivatives in the mitochondria of the cell, conclusions about the cell vitality can be drawn. The results of the measured optic densities are summarized in the following table 1.

(31) TABLE-US-00001 TABLE 1 Proliferation activities of primary (M58) versus Tag58 cells in an undifferentiated or differentiated state, determined by means of a colorimetric assay (EZ4U, Biozol). Listed are the optical densities standard deviation (n = 3). primary cells (M58) Tag58 cells (Tag58) osteogenically osteogenically undifferentiated stimulated undifferentiated stimulated Day 0.419 0.096 0.428 0.051 0.569 0.092 0.595 0.232 3 Day 1.527 0.457 0.823 0.100 2.222 0.166 2.023 0.185 10 Day 2.390 0.181 1.621 0.247 2.090 0.326 1.889 0.145 20

(32) The following findings were made: On day 3 of the osteogenic differentiation there were no significant differences between the parental primary cells (M58) and the Tag58 cells. The values of the optical density tend to be higher in the Tag58 cells, however, reached no significance niveau compared to the parental primary cells.

(33) However, on day 10 of osteogenesis significant differences of the proliferation activity were shown. While osteogenic stimulated primary cells (M58) reduce their proliferation activity on medium-high OD values, the proliferation rates of undifferentiated and differentiated Tag58 cells are not significantly different, however, they are far above the OD values determined for primary cells.

(34) On day 20 of osteogenesis no significant differences between the primary and the Tag58 cells could be detected.

(35) Detection of the Mineralization Potential

(36) By means of the alizarin- or fluorescence-based Osteolmage staining calcium phosphate compositions can be detected (FIG. 3, FIG. 4), which are formed by mesenchymal precusor cells during the osteogenic differentiation process.

(37) By means of the alizarin staining (FIG. 4) it could be detected that in the Tag58 cells the formation of the calcium phosphate precipitates occurs earlier than in the parental primary cells M58. This begins in the immortalized Tag58 cells already after 10 days of the osteogenic stimulation and is clearly detectable after 16 days (d 16) by means of alizarin staining (see FIG. 4). In contrast, a beginning of mineralization in the primary cells M58 was visible only after day 30 of the osteogenic stimulation and was detectable at day 37 (d 37) by means of alizarin staining.

(38) In a synchronized experiment at day 37 (d 37) an intensive Osteolmage staining of the calcium phosphate precipitate in the monolayer of the Tag58 cells could be detected, compared to the primary cells M58 (FIG. 5).

(39) Flow Cytometric Investigations of the Surface Antigen Expression

(40) Various surface antigens such as CD29, CD44, CD73, CD90, CD105 and CD166 have been defined as stem cell markers of mesenchymal stem cell lines. The surface marker expression was examined 10 days after seeding of the cells. CD45 is a leucocyte antigen that is mostly negative in cranium periosteum cells. The parental cells M58 from which the Tag58 cells according to the invention originated, however, show a weak CD45 expression (18.81%, see FIG. 5B). However, the Tag58 cells are almost negative with respect to the CD45 expression. The CD44, CD73, CD105, CD166 expressions showed in the primary as well as in the immortalized cells were relatively comparable (FIG. 5C-F) with the restriction that the percentage of the positive Tag58 for CD73, CD105 and CD166 was slightly lower. Surprisingly, a three-times higher CD146 expression in the Tag58 cells was detected in comparison to the primary cells (see FIG. 5G).

3 Conclusion

(41) The inventor was able to confirm by means of a plurality of experimental evidences that starting from human primary cranium periosteum cells an immortalized periosteum cell line was generated.