METHOD FOR PREPARATION OF IMMORTALIZED STEM CELL LINE AND USE THEREOF
20220282220 · 2022-09-08
Assignee
Inventors
Cpc classification
C12N2740/16043
CHEMISTRY; METALLURGY
C12N2740/15042
CHEMISTRY; METALLURGY
C12N9/1276
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C12N5/0663
CHEMISTRY; METALLURGY
C12N1/04
CHEMISTRY; METALLURGY
C12N2529/00
CHEMISTRY; METALLURGY
A01N1/0284
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
C12N2740/15022
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
C12N13/00
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
International classification
A61K35/28
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
Abstract
The present invention relates to a method for preparation of an immortalized stem cell line that has an immortalizing gene introduced thereinto and retains an expression potential of the introduced gene while being restrained from proliferation so that the immortalized stem cell line can be used as a cell therapeutic agent, and to a use thereof. The immortalized stem cell line of the present invention, which has undergone a radiation process, cannot proliferate while maintaining the expression of the introduced foreign protein at a certain level or higher and as such, can be advantageously used as clinical samples such as various cell therapeutic agents.
Claims
1. A method for preparation of an immortalized stem cell line, comprising: preparing a stem cell line by introducing an immortalizing gene; introducing a gene encoding a foreign protein into the stem cell line; cryopreserving the stem cell line; and irradiating radiation to the cryopreserved stem cell line.
2. The method of claim 1, wherein the immortalizing gene is c-Myc and/or hTERT.
3. The method of claim 1, wherein the foreign protein is selected from the group of growth factors, cytokines or cancer therapeutic proteins, antibodies, and chemokine receptors.
4. The method of claim 1, wherein the immortalized stem cell line is a human embryonic stem cell (hES), a bone marrow stem cell (BMSC), a mesenchymal stem cell (MSC), a human neural stem cell (hNSC), a limbal stem cell, or an oral mucosal epithelial cell.
5. The method of claim 1, wherein the radiation is irradiated so that the absorbed dose to the stem cell line is at least 80 Gy.
6. The method of claim 5, wherein the absorbed dose to the stem cell line satisfies, with respect to irradiation dose, the following range: 80 to 140% of the irradiation dose when the irradiation dose is 0 to 100 Gy; 80 to 140% of the irradiation dose when the irradiation dose is 100 to 200 Gy; and 60 to 140% of the irradiation dose when the irradiation dose is 200 Gy or more.
7. The method of claim 1, wherein the radiation is selected from the group of gamma rays, beta rays, neutron rays, X-rays and electron beams.
8. A pharmaceutical composition comprising the immortalized stem cell line according to the method of claim 1.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises an immortalized stem cell line expressing brain-derived neurotrophic factor (BDNF).
10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition has a preventive or therapeutic effect on neurological diseases.
11. The pharmaceutical composition of claim 10, wherein the neurological disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), cerebral infarction, chronic brain injury, spinal cord injury, Huntington's disease (HD), Rett's disease (RD), ischemic brain disease, stroke and traumatic brain injury, neonatal hypoxic ischemic encephalopathy, and multiple sclerosis.
12. Use of the immortalized stem cell line according to the method of claim 1 as a cell therapeutic agent.
13. A method for treating a disease by administering the immortalized stem cell line according to the method of claim 1 in a therapeutically effective amount to a subject in need thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] The above and other aspects, features, and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing embodiments thereof in detail with reference to the accompanying drawings, in which:
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[0084] X-axis: culture period; and
[0085] Y-axis: cumulative population doubling level (PDL).
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[0087] imMSC: immortalized MSC;
[0088] MSC: non-immortalized MSC;
[0089] X-axis: culture period; and
[0090] Y-axis: cumulative population doubling level (PDL).
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[0094] X-axis: culture period; and
[0095] Y-axis: cumulative population doubling level (PDL).
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[0097] X-axis: culture period; and
[0098] Y-axis: cumulative population doubling level (PDL).
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[0118] X-axis: number of BM102 cells; and
[0119] Y-axis: proliferation rate of C6 cells.
BEST MODE FOR CARRYING OUT THE INVENTION
[0120] In an aspect, the present invention relates to a method for preparation of an immortalized stem cell line, the method comprising: preparing a stem cell line by introducing an immortalizing gene, introducing a gene encoding a foreign protein into the stem cell line, cryopreserving the stem cell line, and irradiating radiation to the cryopreserved stem cell line.
[0121] As an embodiment of the present invention, the immortalizing gene is c-Myc and/or hTERT.
[0122] As an embodiment of the present invention, the foreign protein is selected from the group of growth factors, cytokines or cancer therapeutic proteins, antibodies, and chemokine receptors.
[0123] As an embodiment of the present invention, the immortalized stem cell line is a human embryonic stem cell (hES), a bone marrow stem cell (BMSC), a mesenchymal stem cell (MSC), a human neural stem cell (hNSC), a limbal stem cell, or an oral mucosal epithelial cell. As an embodiment of the present invention, the radiation is irradiated so that the absorbed dose to the stem cell line is at least 80 Gy, and more specifically, the absorbed dose satisfies the range of: 80 to 140% of the irradiation dose when the irradiation dose is 0 to 100 Gy; 80 to 140% of the irradiation dose when the irradiation dose is 100 to 200 Gy; and 60 to 140% of the irradiation dose when the irradiation dose is 200 Gy or more.
[0124] As an embodiment of the present invention, the radiation is selected from the group of gamma rays, beta rays, neutron rays, X-rays and electron beams.
[0125] In another aspect, the present invention relates to a pharmaceutical composition comprising an immortalized stem cell line prepared by the above method.
[0126] As an embodiment of the present invention, the pharmaceutical composition comprises an immortalized stem cell line expressing brain-derived neurotrophic factor (BDNF).
[0127] As an embodiment of the present invention, the pharmaceutical composition has a preventive or therapeutic effect on neurological diseases, and the neurological disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), cerebral infarction, chronic brain injury, spinal cord injury, Huntington's disease (HD), Rett's disease (RD), ischemic brain disease, stroke and traumatic brain injury, neonatal hypoxic ischemic encephalopathy, and multiple sclerosis.
[0128] In yet another aspect, the present invention relates to a use of an immortalized stem cell line prepared by the above method as a cell therapeutic agent.
[0129] In yet another aspect, the present invention relates to a method for treating a disease by administering an immortalized stem cell line prepared by the above method in a therapeutically effective amount to a subject in need thereof.
MODE FOR CARRYING OUT THE INVENTION
[0130] Hereinafter, the present invention is described in detail with reference to the following Examples. However, the following Examples are only intended to illustrate the present invention, and the present invention is not limited thereto.
Example 1. Preparation of Immortalized Mesenchymal Stem Cells (MSCs)
Example 1.1. Preparation of Lentiviral Vector Containing Immortalizing Gene
[0131] To immortalize MSCs, lentiviral vectors containing immortalizing genes c-Myc and/or hTERT were prepared. In this case, a gene construct expressing a tTA protein was also inserted to use a Tet-off system.
[0132] First, a pBD lentiviral vector was prepared by substituting an EF promoter in an expression cassette of a pWPT vector (Bioneer, synthesized) with a CMV promoter. The c-Myc gene (SEQ ID NO: 7) was inserted into the pBD lentiviral vector so that the expression could be regulated by the CMV promoter. The vector prepared above was named pBD-1.
[0133] Meanwhile, the hTERT gene (SEQ ID NO: 8) was inserted into the pBD lentiviral vector so that the expression could be regulated by the CMV promoter. A gene having resistance to zeomycin (ZeoR; SEQ ID NO: 12) was inserted thereinto so that the expression could be regulated by an RSV promoter. The vector prepared above was named pBD-2.
[0134] In addition, the tTA (tetracycline transactivator) gene (SEQ ID NO: 9) was inserted into the pBD lentiviral vector so that the expression could be regulated by the CMV promoter. The vector prepared above was named pBD-3.
Example 1.2. Production of Lentivirus Containing Immortalizing Gene
[0135] Using the lentiviral vectors prepared in Example 1.1, lentiviruses containing an immortalizing gene were produced by the following method.
[0136] First, Lenti-X cells (Clontech Laboratories, USA) were cultured in a 150 mm dish using DMEM medium containing 10% fetal bovine serum. Meanwhile, the lentiviral vectors were extracted and quantified from DH5a E. coli cells using the EndoFree Plasmin Maxi Kit (Qiagen, USA).
[0137] After the cultured Lenti-X cells were washed with PBS, 3 ml of TrypLE™ Select CTS™ (Gibco, USA) was added. After leaving the cells at 37° C. for about 5 minutes, it was confirmed that the cells were detached. The detached cells were neutralized by adding 7 ml of DMEM medium containing 10% fetal bovine serum. The neutralized cells were collected in a 50 ml tube and centrifuged at 1,500 rpm for 5 minutes. The cells were re-suspended by removing the supernatant and adding 10 ml of DMEM culture medium containing 10% fetal bovine serum.
[0138] Suspended cells were counted with a hematocytometer, and then aliquoted into 1.2×10.sup.7 cells in a 150 mm dish. When the aliquoted cells were cultured to reach about 90% confluence, the cells were transduced with a mixture of 12 μg of lentiviral vector, 12 μg of psPAX (Addgene; gag-pol expression, packaging plasmid) and 2.4 μg of pMD.G plasmid (Addgene; VSVG expression, envelope plasmid). Lipofectamine (Invitrogen, USA) and Plus Reagent (Invitrogen, USA) were used to aid in transduction. After 6 hours of transduction, the medium was exchanged with DMEM containing 10% fetal bovine serum. After the cells were cultured for an additional 48 hours, the supernatant was collected.
[0139] The obtained supernatant was mixed with a lentivirus concentration kit (Lenti-X concentrator, Clontech Laboratories, USA), and then cultured at 4° C. overnight. A virus was obtained by centrifuging the supernatant under the conditions of 4° C. and 4,000 rpm for 2 hours, and the virus was re-suspended in 0.5 ml of DMEM without FBS.
Example 1.3. Preparation of Immortalized Mesenchymal Stem Cells
[0140] Immortalized MSCs were prepared using the lentiviruses containing the immortalizing genes produced in Example 1.2.
[0141] First, bone marrow-derived MSCs were prepared by the following method. Specifically, a bone marrow aspirate was obtained from the iliac crest of a healthy donor. The bone marrow aspirate was mixed with 20 IU/ml heparin in a sterile container to inhibit coagulation. After the bone marrow mixture was centrifuged under the conditions of 4° C. and 739 G for 7 minutes, the supernatant was removed and was mixed with 10-fold volumes of sterilized water. A pellet of cells was obtained by centrifuging the mixture again under the same conditions. The obtained pellet was suspended in
[0142] DMEM-low glucose (11885-084, Gibco, USA) medium containing 20% fetal bovine serum and 5 ng/ml b-FGF (100-18B, Peprotech, USA) and aliquoted into a culture flask. After the aliquoted one was cultured under the conditions of 37° C., and 5% CO.sub.2 for 24 to 48 hours, the medium was exchanged with a new medium. The cultured one was subcultured while exchanging the medium with a new medium every 3 to 4 days, and after 2 weeks of culture, it was checked whether MSCs were present using a fluorescence cell analyzer.
[0143] The prepared MSCs were infected with the pBD-1 lentivirus produced in Example 1.2 at 100 MOI using Retronectin (Clontech Laboratories, USA). As a result, the cell proliferation rates of MSCs containing c-myc and MSCs not containing c-myc are shown in
[0144] In addition, cells infected with the lentivirus containing c-Myc were infected with the pBD-2 lentiviral vector containing hTERT at 100 MOI. After infection, 500 μg/ml of zeomycin was added to the culture medium of the stabilized cells to select pBD-2 lentivirus-infected cells.
[0145] In this regard, as shown in
[0146] In addition, cells infected with the pBD-1 and/or pBD-2 were infected with the pBD-3 lentiviral vector containing tTA at 100 MOI. After infection, 1 μg/ml of puromycin was added to the culture medium of the stabilized cells to select pBD-3 lentivirus-infected cells.
[0147] Example 2. Preparation of Lentivirus Containing Foreign Gene
Example 2.1. Preparation of Lentivirus Containing BDNF Gene
[0148] A BDNF gene (SEQ ID NO: 2) was inserted into the pBD lentiviral vector prepared in Example 1.1.
[0149] First, in order to confirm the effect of a promoter on BDNF expression, a lentiviral vector was prepared to express the BDNF gene using a CMV promoter and a TRE promoter, respectively and MSCs expressing the BDNF gene were prepared using the lentiviral vector. As a result, it was confirmed that in the case of a cell line to which the CMV promoter was applied, the MSCs expressing BDNF were morphologically changed during long-term subculture, and the expression rate of BNDF decreased as the subculture progressed.
[0150] Accordingly, the BNDF gene was inserted into each pBD vector so that expression was regulated by the TRE promoter. The TRE promoter can regulate the expression of the gene linked to the promoter depending on whether doxycycline is added or not.
[0151] In addition, using the lentiviral vectors pBD-4 and pBD-5 containing the BDNF gene prepared above, lentiviruses were produced in the same manner as described in Example 1.2. The produced lentivirus was prepared at a concentration of 4.7×10.sup.6 copies/ml (pBD-4) and 1.4x10.sup.12 copies/ml (pBD-5), respectively.
Example 2.2. Preparation of Lentivirus Containing TRAIL and CD Genes
[0152] Lentiviruses containing TRAIL and CD genes were prepared according to the contents described in Korean Patent No. 10-1985271. TRAIL gene (SEQ ID NO: 4) and CD gene (SEQ ID NO: 6) were inserted into the pBD lentiviral vector prepared in Example 1.1. In this case, the inserted TRAIL and CD genes were linked to an internal ribosome entry site (IRES) such that expression is regulated by the TRE promoter. The IRES is a ribosome binding site, allowing translation to start even without a 5′-cap structure, so that two proteins can be expressed in one mRNA. Meanwhile, the TRE promoter can regulate the expression of the gene linked to the promoter depending on whether doxycycline is added or not.
[0153] The vector into which the TRAIL and CD were inserted was named pBD-6, and using this, a lentivirus was produced in the same manner as described in Example 1. The produced lentivirus was prepared at a concentration of 7.6×10.sup.8 TU/ml.
Example 3. Preparation of Immortalized Stem Cells Transfected With Lentivirus Containing Foreign Gene
Example 3.1. Preparation of MSCs Transfected With Lentivirus Containing BDNF Gene
3.1.1. Preparation of BM102 Cell Line
[0154] The immortalized MSC prepared in Example 1.3 was infected with the lentivirus containing the BDNF gene produced in Example 2.1 at 100 MOI to prepare cells expressing the BDNF gene. Here, the lentivirus produced by the pBD-1 vector into which among immortalizing genes c-Myc was introduced was used. The transduced cells were cultured in a medium added with 2 μg/ml doxycycline (631311, Clontech, USA) to suppress the expression of BDNF protein during culture.
[0155] The cells were cultured to form colonies using a clone select imager. After numbering each well inoculated with cells in a 96-well plate, the presence or absence of BDNF protein expression was confirmed with the human BDNF DuoSet ELISA kit (R&D systems, DY248, USA) among about 200 clones that confirmed colony formation. Among the cells, for immortalized MSCs prepared by lentivirus introduced with pBD-4, selected were clones #10, #22, #28, #65, #110, #116, #126, #596, #669 and #741 expressing BDNF protein of 1.5 ng/ml or more following doxycycline removal. Thereafter, clones #28 and #110 expressing BDNF protein at 0.5 ng/ml or less upon doxycycline treatment were selected (
[0156] As a result of confirming the cell proliferation ability of the selected clones, clone #28 showing a stable proliferation pattern and the best BDNF protein expression level was named BM102 cell line and deposited as a patent strain (Korea Research Institute of Bioscience and Biotechnology, KCTC 13876BP).
3.1.2. Preparation of BM01A Cell Line
[0157] In addition, in the same manner as in 3.1.1 above, immortalized MSCs infected with lentiviruses introduced with pBD-1 and pBD-2 were prepared. The method for preparation of the cell line was according to the method described in Korean Patent No. 10-2074336. After virus infection, after stabilizing the cells, 500 μg/ml of G418 was added to the culture medium to select cells infected with pBD-5 lentivirus into which both c-Myc and hTERT genes were introduced. The selected cells were cultured in a medium added with 1 μg/ml doxycycline (631311, Clontech, USA) to suppress the expression of BDNF protein during culture.
[0158] The selected cells were cultured to form colonies. By confirming the presence or absence of BDNF protein expression with human BDNF DuoSet ELISA kit (R&D systems, DY248, USA), among the clones #1 to #50 formed, clones #10, #12, #14, #18, #20, #22, #23, #26, #29 and #41 expressing BDNF protein were selected, and clones #14, #22, #23 and #41 not expressing BDNF protein upon doxycycline treatment were reselected (
Example 3.2. Preparation of MSCs Transfected With Lentiviruses Containing TRAIL and CD Genes
[0159] According to the method described in Korean Patent No. 10-1985271, the immortalized MSC prepared in Example 1-3 was infected with a lentivirus containing the TRAIL and CD genes produced in Example 2-2 to prepare cells expressing TRAIL and CD genes. Infection was performed in the same manner as described in Examples 1-3. After infection, 1 μg/ml of doxycycline (631311, Clontech, USA) was added to the stabilized cell culture medium and cultured in a state in which the expression of TRAIL and CD was suppressed. After the cells were stabilized, the cells were cultured for 72 hours in a culture medium without doxycycline to induce expression of TRAIL and CD, and FACS was performed with the cells to select cells expressing TRAIL on the cell surface.
[0160] Specifically, the cells infected with the lentivirus containing the TRAIL and CD genes were aliquoted to 5×10.sup.5 per FACS tube, and the supernatant was removed by centrifugation at 4° C. at 1,500 rpm for 5 minutes. To this, 1 ml of FACS buffer (PBS containing 2% fetal bovine serum) was added to resuspend the cells, and centrifuged under the same conditions to remove the supernatant. After performing the above washing procedure once more, the cells were resuspended in 1 ml of FACS buffer. A mixture of 0.3 μl LIVE/DEAD® Fixable Near-IR Dead Cell Stain (Life Technologies-Molecular Probes, USA) and 5 μl APC anti-human CD253 antibody, which is an anti-TRAIL antibody, (BioLegend, Cat#. 308210, USA) added in 200 μl FACS buffer was added to the resuspended cells, and reacted at 4° C. for 30 minutes. After the reaction, the cells were washed twice in the same manner as above, and the supernatant was removed. 300 μl of fixing buffer (PBS containing 2% formaldehyde and 1% fetal bovine serum) was added to the washed cells, and the cells were left at 4° C. for at least 15 minutes. The cells were analyzed with a FACS instrument (LSRFortessa, BD biosciences, USA), and cells expressing TRAIL were selected and cultured to form colonies.
[0161] A cell line was established by culturing monoclonal cells from the formed colonies, and this was named BM-03.
Example 4. Irradiation Test for Immortalized Stem Cell Line
[0162] In order to use each immortalized MSC cell line prepared in Example 3 as a clinical sample, a irradiation step was additionally performed to maintain the expression level of the therapeutic protein without cell proliferation, and through this, a suitable irradiation (absorbed) dose was confirmed.
[0163] First, the immortalized MSC cell line formulated and cryopreserved in a liquid nitrogen tank was transported using a mobile cartridge prepared for irradiation. In this case, the cryopreserved was maintained using dry ice.
[0164] Irradiation was performed on the cryopreserved immortalized MSC cell line. Irradiation was carried out using a known method and device, and for gamma rays, a low level gamma ray irradiation device or a high level gamma ray irradiation device (MDS Nordion, Canada) was used, and for X-rays, X-RAD 320 X-RAY IRRADIATOR (Seoul National University Graduate School of Convergence Science and Technology) or RS1800Q (Rad Source technologies, Inc) was used. Even during irradiation, the temperature inside the mobile cartridge was maintained at a temperature that could sustain the cryopreserved by dry ice, and the irradiation-completed MSC cell line was stored again in a liquid nitrogen tank.
[0165] In order to confirm the irradiation dose, the radiation absorbed dose was measured for each dose using a separate alanine dosimeter, and the absorbed dose of the immortalized MSC cell line could be calculated from the above results.
[0166] After thawing 3 vials for each specimen of the irradiation-completed MSC cell line for about 2 minutes in a 37° C. constant-temperature water bath, 9 mL of PBS was added and centrifuged for 5 minutes at 4° C. and 1,500 rpm, and then the supernatant was removed, and the cells was suspended in DMEM-low glucose (11885-084, Gibco, USA) medium containing 10% FBS (16000-044, Gibco, USA) and 10 ng/mL b-FGF (100-18B, Peprotech, USA). The number of suspended cells was measured with a hematocytometer, and then the experiment was performed.
Experimental Example 1. Confirmation of Surface Antigen Protein Expression in BM102 Cell Line
[0167] The expression of surface antigen proteins of the bone marrow-derived MSC before gene insertion and the BM102 cell line in which the BDNF gene was inserted was analyzed using a human MSC analysis kit (Stemflow.sup.Tm, Cat No. 562245, BD). The experiment was performed according to the manual included in each kit, and the experimental results are shown in
[0168] As a result, as shown in
Experimental Example 2. Confirmation of Proliferation Rate of BM102 Cell Line Before Irradiation
[0169] The proliferation rate of the BM102 cell line established in Example 3.1 was confirmed. 0.2×10.sup.6 to 0.8×10.sup.6 cells of the BM102 cell line were inoculated into a T175 flask and then cultured for 3 or 4 days with or without the addition of doxycycline, and during which, PDL (population doubling level) and cell viability were measured. PDL was calculated by the formula “PDL=X+3.222 (logY−logI)”, where X denotes the initial PDL, I denotes the initial number of cells inoculated into the flask, and Y denotes the final number of cells. The proliferation rates of the established cell lines are shown in
[0170] As a result, as shown in
Experimental Example 3. Morphological Confirmation of BM102 Cell Line According to Subculture
[0171] In order to confirm the morphological change according to the subculture of the BM102 cell line established in Example 3.1, in Experimental Example 2, the proliferation rate of the BM102 cell line was confirmed, and the cells were photographed using microscope, and the morphology of the BM102 cells is shown in
[0172] As a result, as shown in
Experimental Example 4. Confirmation of Expression of BDNF Protein in BM102 Cell Line Before Irradiation
[0173] The expression of the BDNF protein in the BM102 cells established in Example 3.1 was confirmed by ELISA analysis. Specifically, the expression level of the BDNF protein was confirmed with the human BDNF DuoSet ELISA kit. The experiment was performed according to the manual included in each kit. The expression levels of the BDNF protein whose expression was induced for 48 hours from about 1×10.sup.5 cells in a medium with the addition or removal of doxycycline were shown in
[0174] As a result, as shown in
TABLE-US-00001 TABLE 1 Target protein Expression level BDNF 740 ng/10.sup.5 cells
[0175] As a result, as shown in Table 1, it was confirmed that about 740 ng/10.sup.5 cells of BDNF protein were expressed in the BM102 cell line before irradiation.
Experimental Example 5. Confirmation of BDNF Transgene in BM102 Cell Line
[0176] PCR was performed to confirm whether a gene was introduced into the BM102 cell line prepared in Example 3.1. Specifically, the BM102 cell line was transferred to a 15 ml tube containing 9 ml of PBS and then cell down was performed at 1,500 rpm for 5 minutes. After the PBS was completely removed, the pellet was suspended in 200 μl of PBS and then transferred to a 1.5m1 tube. Thereafter, gDNA was prepared using NucleoSpin® Tissue (MN, 740952.250), a mixture was prepared as shown in Table 2 below, and then PCR was performed with the steps shown in the Table 3 below. In this case, 100 ng of BM102 plasmid DNA was added as a positive control, and 1μl of purified water was added as a negative control. The BM102 plasmid DNA can be separated and purified according to the method described in Korean Patent Application Laid-Open No. 2017-0093748.
TABLE-US-00002 TABLE 2 Forward primer (SEQ ID NO: 10) 1 (10 pmol/
, Cosmogenetech synthesis) Reverse primer (SEQ ID NO: 11) 1
(10 pmol/
, Cosmogenetech synthesis) Specimen (100 ng/
) 1
Purified water 17
Total volume 20
TABLE-US-00003 TABLE 3 Step Temperature Time Number 1 95° C. 5 minutes 1 2 95° C. 30 seconds 35 62° C. 30 seconds 72° C. 40 seconds 3 72° C. 7 minutes 1 4 4° C. Unlimited 1
[0177] A 1% (v/v) agarose gel was put into an electrophoresis kit. 10 μl of DNA Size Marker was loaded into the first well, and from the next well, each 10 μl was loaded in the order of a negative control, a positive control, and 3 BM102 specimens. Thereafter, electrophoresis was performed at 100 V for 20 minutes, a gel photograph was taken, and the results are shown in
Experimental Example 6. Confirmation of Transcriptome Changes in BM102 Cell Line
[0178] In the BM102 cell line prepared in Example 3.1, transcriptome sequencing analysis was performed to confirm the change in gene expression caused by BDNF expression depending on whether doxycycline was treated.
[0179] The sequencing analysis was commissioned to Macrogen to secure the results. The test was performed according to NovaSeq 6000 System User Guide Document #1000000019358 v02 (illumina) using NovaSeq 6000 S4 Reagent Kit (illumina), and analysis was performed through NovaSeq sequencer and 1000000019358 v02 software (illumina). Transcriptomes that change according to the removal of doxycycline in the BM102 cell line are listed in
[0180] As a result, as shown in
[0181] In addition, as shown in
[0182] Additionally, according to the removal of doxycycline, in the BM102 cell line, transcriptome changes of micro RNA genes were also confirmed, and specifically, it was confirmed that the expression of MiR4444-1, MiR4444-2 and MiR1244-1 was increased, and the expression of MiR1244-4, MiR319I and MiRLET7D was decreased (
Experimental Example 7. Irradiation Test for BM102 Cell Line
[0183] For the BM102 cell line, a irradiation test using gamma rays and X-rays was performed in the same manner as in Example 4, and gamma rays and X-rays were used as the types of radiation to be irradiated.
[0184] First, after primary irradiation with gamma rays at irradiation doses of 80, 100, 120, and 140 Gy, the absorbed dose, colony formation or not, cell number and titer of the BM102 cell line were confirmed, and after secondary irradiation with gamma rays at doses of 200 and 300 Gy, the absorbed dose, colony formation or not, cell number and titer were respectively confirmed in the same manner.
[0185] The results are shown in Table 4 below.
TABLE-US-00004 TABLE 4 Irradia- Primary experiment Secondary experiment tion Absorbed Colony Titer Absorbed Colony Titer dose dose Formation ng/5 × 10.sup.5/ Suitable/ dose Formation ng/5 × 10.sup.5/ Suitable/ (Gy) (Gy) or not 48 hrs unsuitable (Gy) or not 48 hrs unsuitable 0 Gy — — 1259.8 — — — — — 80 Gy 79.5 ◯ 960.2 unsuitable — — — — 90 Gy — — — — — — — 100 Gy 99.6 X 826.6 suitable — — — — 110 Gy — — — — — — — — 120 Gy 121.2 X 963.3 suitable — — — — 130 Gy — — — — — — — — 140 Gy 137.6 X 849.9 suitable — — — — 200 Gy — — — — 219.7 X 400 or more suitable 300 Gy — — — — 322.2 X 400 or more suitable
[0186] In addition, the absorbed dose, colony formation or not and titer were confirmed by first irradiation with irradiation doses of 100, 200, 300, 400, and 500 Gy, respectively, using X-rays, and the results are shown in Table 5 and
TABLE-US-00005 TABLE 5 BM102 X-ray irradiation (primary) Irradiation Absorbed Colony dose dose Formation Titer Suitable/ (Gy) (Gy) or not ng/10.sup.5 unsuitable 100 Gy 62.5 ◯ 122.4 unsuitable 300 Gy 187.5 X 121.3 suitable 400 Gy 250 X 120 suitable 500 Gy 312.5 X 107.7 suitable
TABLE-US-00006 TABLE 6 BM102 X-ray irradiation (secondary) After Irradiation Absorbed thawing Colony Titer dose dose Cell Formation ng/10.sup.5/ Suitable/ (Gy) (Gy) viability or not 60 hr unsuitable 0 Gy — 96.8 NA 325.1 NA 50 Gy 41.3 96.7 X 247.5 suitable 100 Gy 83.1 95.1 X 252.1 suitable 200 Gy 155.5 95.6 X 249.4 suitable 300 Gy 232.1 98.9 X 250.7 suitable 400 Gy NA* 96.4 X 240.7 suitable 500 Gy NA* 96.5 X 191.7 suitable
Experimental Example 7.1. Colony Formation or Not of the Irradiated BM102 Cell Line
[0187] For the BM102 cell line, to check whether colonies are formed after irradiating gamma rays and X-rays in the above ranges, respectively, the cells were aliquoted into 12 wells per vial so that there were 5×10.sup.5 cells in a well in a 6-well plate, and a total of two 6-well plates were aliquoted. The plate was shaken to spread the cells evenly, the medium was changed every 3 or 4 days in a 37° C., 5% CO.sub.2 incubator, and it was visually observed whether colonies were formed using a microscope. As a result, as shown in Table 4 above, upon irradiating gamma rays, colonies were formed when the irradiation dose was 80 Gy, and it was confirmed that colony formation was not made in a range higher than that. When colonies were formed, the radiation absorbed dose of the cell line was confirmed to be 79.5 Gy, and the maximum absorbed dose confirmed experimentally was about 322 Gy.
[0188] In addition, as a result of X-ray irradiation, it was also confirmed whether colonies were formed. As shown in Tables 5 and 6 above, in the primary experiment, when the irradiation dose was 100 Gy, colonies were formed, confirming that it was not suitable for use as a clinical sample, and colonies were not formed in a range beyond that. When colonies were formed, the absorbed dose of the cell line was found to be 62.5 Gy.
[0189] On the other hand, as confirmed in the secondary X-ray irradiation experiment, when irradiated with 100 Gy, the absorbed dose was measured to be 83.1 Gy. At this time, no colonies were formed. That is, even when irradiated with the same irradiation dose, there may be differences in absorbed dose, and it was confirmed that when the dose absorbed into the cells was 62.5 Gy, it was not suitable for use as a clinical sample, but it can be used in the case of 83.1 Gy. Therefore, when radiation in a range exceeding about 80 Gy is absorbed based on the absorbed dose, it can be predicted that the cell line of the present invention can be used as a clinical sample.
Experimental Example 7.2. Results of Cell Number Measurement of the Irradiated BM102 Cell Line
[0190] In order to test whether the colony was formed, a cell number measurement test was performed using the inoculated cells. For the BM102 cell line irradiated with gamma rays in Experimental Example 7, on each 7, 14, 28, 35, 49, and 63 days based on the cell thawing day, the attached cells were detached using trypsin, and then stained with trypan blue and the total number of cells and the number of viable cells were counted in a hematocytometer.
[0191] As a result, as shown in Table 7 below and
TABLE-US-00007 TABLE 7 Primary test (Dose) Secondary test (Dose) Time Item 0 Gy 80 Gy 100 Gy 120 Gy 140 Gy 200 Gy 300 Gy Day 0 Number of 5.0 5.0 5.0 5.0 5.0 5.0 5.0 cells (× 10.sup.5) Viability (%) 91.5 91.9 93.9 93.5 94.9 90.2 93.9 Day 7 Number of 22.75 5.09 3.91 3.19 2.95 4.39 4.55 cells (× 10.sup.5) Viability (%) 93.1 93.9 97.7 98.0 97.6 90.5 91.6 Day 14 Number of 20.13 4.83 3.70 3.06 2.92 4.03 3.69 cells (× 10.sup.5) Viability (%) 84.5 88.2 87.5 90.2 91.8 89.2 89.5 Day 21 Number of — 3.13 3.32 2.88 2.21 3.57 3.45 cells (× 10.sup.5) Viability (%) — 81.3 84.3 83.6 74.1 89.9 88.5 Day 28 Number of — 2.81 2.77 2.44 2.08 3.40 3.37 cells (× 10.sup.5) Viability (%) — 86.8 90.6 89.6 87.2 88.3 89.1 Day 35 Number of — 2.69 2.62 2.53 2.11 2.93 2.88 cells (× 10.sup.5) Viability (%) — 85.6 84.3 87.5 85.9 85.4 84.3 Day 49 Number of — 2.33 2.26 2.14 1.70 2.57 2.56 cells (× 10.sup.5) Viability (%) — 84.8 83.7 83.9 74.6 80.9 79.6 Day 63 Number of — 2.25 2.23 1.98 1.55 2.48 2.47 cells (× 10.sup.5) Viability (%) — 83.2 82.1 81.5 71.6 79.4 78.6
[0192] In addition, for the cell lines subjected to irradiation experiment using X-rays, as a result of measuring the number of cells at each 14, 28, 42, 56, and 70 days after the cell line was thawed, as shown in Table 8 below and
TABLE-US-00008 TABLE 8 Dose Time Item 100 Gy 300 Gy 400 Gy 500 Gy Day 0 Number of cells (×10.sup.5) 5.0 5.0 5.0 5.0 Viability (%) 92.5 93.8 94.3 95.5 Day 14 Number of cells (×10.sup.5) 2.01 1.05 0.71 0.83 Viability (%) 92.0 81.7 88.3 88.5 Day 28 Number of cells (×10.sup.5) 7.79 0.85 0.74 0.73 Viability (%) 79.9 71.2 63.4 66.9 Day 42 Number of cells (×10.sup.5) — 0.99 0.89 0.64 Viability (%) — 77.5 71.0 60.7 Day 56 Number of cells (×10.sup.5) — 0.96 0.94 0.66 Viability (%) — 74.0 76.0 69.7 Day 70 Number of cells (×10.sup.5) — 0.94 0.85 0.64 Viability (%) — 73.8 74.8 69.9
Experimental Example 7.3. Confirmation of BDNF Expression in the Irradiated BM102 Cell Line
[0193] Some of the BM102 cells irradiated in Experimental Example 7 were aliquoted into a total of 1 well per vial so that 1×10.sup.5 or 5×10.sup.5 cells were placed in a well in a 12-well plate for titer test. The plate was shaken to spread the cells evenly, and it was cultured for 48 hours or 60 hours in a 37° C., 5% CO.sub.2 incubator.
[0194] To measure the expression level of BDNF, a supernatant was obtained from the culture medium, centrifuged at 5,000 rpm at 4° C. for 5 minutes, and the supernatant was divided into 4 e-tubes by 200 μL each and cryopreserved at −80° C. The expression level of BDNF protein was analyzed using a BDNF assay kit (DBD00, R&D systems, USA).
[0195] In the case of the BM102 cell line irradiated with gamma rays, as shown in Table 4 and
Experimental Example 8. Irradiation Test for BM01A Cell Line
[0196] In the same manner as in Experimental Example 7, a irradiation test was performed on the BM01A cell line. Gamma rays were used as the type of radiation to be irradiated, and after primary irradiation with gamma rays at irradiation doses of 90, 100, 110, and 120 Gy, the absorbed dose, colony formation or not, cell number and titer were confirmed, and after secondary irradiation with gamma rays at doses of 140, 160 and 180 Gy, the absorbed dose, colony formation or not, cell number and titer were respectively confirmed in the same manner. The results are shown in Table 9 below.
TABLE-US-00009 TABLE 9 Irradia- Primary experiment Secondary experiment tion Absorbed Colony Titer Absorbed Colony Titer dose dose Formation (ng/5 × 10.sup.5/ Suitable/ dose Formation (ng/5 × 10.sup.5/ Suitable/ (Gy) (Gy) or not 48 hrs) unsuitable (Gy) or not 48 hrs) unsuitable 0 Gy — — 540.7 — — — 611.3 — 90 Gy 95.6 X 1238.6 suitable — — — — 100 Gy 108 X 896.1 suitable — — — — 110 Gy 120.6 X 909 suitable — — — — 120 Gy 131.1 X 1063.4 suitable — — — — 140 Gy — — — — 154.1 X 1018.3 suitable 160 Gy — — — — 173.8 X 870 suitable 180 Gy — — — — 191.4 X 993.4 suitable
Experimental Example 8.1. Colony Formation or Not of the Irradiated BM01A Cell Line
[0197] The BM01A cell line was aliquoted in a 6-well plate at the number of 3×10.sup.5 cells/well, and after culturing in the same manner as in Experimental Example 7.1, colony formation or not was confirmed in the medium.
[0198] As a result, as shown in Table 9 above, colonies were not formed at all irradiation doses in the range of 90 to 120 Gy and 140 to 180 Gy, and it was confirmed that the minimum absorbed dose at which colonies were not formed was 95.6 Gy and the maximum absorbed dose was 191.4 Gy.
Experimental Example 8.2. Results of Cell Number Measurement of the Irradiated BM01A Cell Line
[0199] In the same manner as in Experimental Example 7.2., a test for measuring the number of cells in the BM01A cell line irradiated with gamma rays was performed.
[0200] On 7, 14, 28, 35, and 42 days based on the cell thawing day, the attached cells were detached using trypsin, and then stained with trypan blue and the total number of cells and the number of viable cells were counted in a hematocytometer.
[0201] As a result, as shown in Table 10 below and
TABLE-US-00010 TABLE 10 Primary test (Dose) Secondary test (Dose) Time Item 0 Gy 90 Gy 100 Gy 110 Gy 120 Gy 0 Gy 140 Gy 160 Gy 180 Gy Day 0 Number of 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 cells (× 10.sup.5) Viability 92.9 99.7 99.0 99.4 87.7 90.2 91.0 89.4 92.7 (%) Day 7 Number of 9.44 1.66 1.57 1.32 1.70 24.03 2.44 2.42 2.86 cells (× 10.sup.5) Viability 88.8 89.5 88.5 75.0 87.1 86.1 71.7 73.0 75.2 (%) Day 14 Number of — 1.18 0.89 1.14 0.98 — 0.62 0.85 1.04 cells (× 10.sup.5) Viability — 46.4 48.3 56.3 53.1 — 26.3 29.7 36.0 (%) Day 21 Number of — 0.23 0.30 0.22 0.23 — 0.14 0.08 0.11 cells (× 10.sup.5) Viability — 39.7 38.6 29.2 29.0 — 3.6 2.5 3.5 (%) Day 28 Number of- — 0.14 0.08 0.09 0.08 — 0.04 0.07 0.05 cells (× 10.sup.5) Viability — 26.8 13.4 16.4 12.6 — 1.9 2.9 2.7 (%) Day 35 Number of — 0.04 0.06 0.03 0.04 — 0.03 0.03 0.02 cells (× 10.sup.5) Viability — 6.3 12.0 4.9 5.9 — 4.0 3.7 2.3 (%) Day 42 Number of — 0.01 0.02 0.01 0.02 — 0.01 0.03 0.02 cells (× 10.sup.5) Viability — 3.2 4.2 2.1 3.8 — 0.6 2.3 1.6 (%)
Experimental Example 8.3. Confirmation of BDNF Expression Level in the Irradiated BM01A Cell Line
[0202] For titer test of the BM01A cell line irradiated in Experimental Example 8, cells were cultured and cryopreserved in the same manner as in Experimental Example 7.3. The BDNF expression level of BM01A was analyzed using a BDNF assay kit (DY248, R&D systems, USA).
[0203] As a result, as shown in Table 9 and
Experimental Example 9. Irradiation Test for BM03 Cell Line
[0204] For the BM03 cell line, which is an immortalized stem cell expressing TRAIL and CD, a irradiation test was performed in the same manner as in Experimental Example 7. The irradiation test of the BM03 strain utilized gamma rays, and the results were confirmed by irradiating low-level and high-level gamma rays, respectively.
[0205] For low-level gamma rays, BM03 (BM03-P005, 1×10.sup.7 cells/ml/vial) cryopreserved after filling in a vial was irradiated using a low-level irradiator at the Advanced Radiation Technology Institute of Korea Atomic Energy Research Institute, and irradiation doses of 0, 10, 20, 30, 40, 50, 60, 70 Gy were respectively irradiated and the absorbed dose, colony formation or not, cell number and titer were confirmed as shown in Table 11 below.
TABLE-US-00011 TABLE 11 Low level Irradia- Primary experiment tion Absorbed Colony Titer (5x10.sup.(a+2)) dose dose* formation TRAIL (a) CD::UPRT (a) Suitable/ (Gy) (Gy) or not Day 0 Day 2 Day 7 Day 14 Day 0 Day 2 Day 7 Day 14 unsuitable 0 Gy (0 Gy) — 8.92 9.26 8.82 9.20 8.64 9.17 8.66 9.08 — 10 Gy 10 Gy ◯ 8.62 9.34 8.76 8.98 8.38 9.20 8.61 8.81 unsuitable 20 Gy 20 Gy ◯ 8.65 9.19 8.60 8.85 8.40 9.06 8.43 8.71 unsuitable 30 Gy 30 Gy ◯ 9.13 9.13 8.81 8.45 8.73 9.01 8.71 8.45 unsuitable 40 Gy 40 Gy ◯ 8.79 8.93 8.62 8.35 8.63 8.78 8.50 8.23 unsuitable 50 Gy 50 Gy ◯ 8.60 8.71 8.76 8.46 8.40 8.70 8.68 8.41 unsuitable 60 Gy 60 Gy X 8.65 8.94 9.04 8.59 8.42 8.96 8.96 8.53 unsuitable 70 Gy 70 Gy X 9.06 9.12 8.94 8.58 8.75 9.13 8.78 8.56 suitable
[0206] In addition, in the same manner, a high-level gamma-ray irradiation test was performed, and for high-level gamma rays, the primary test at doses of 0, 30, 40, 60, 70 Gy, and the secondary test at doses of 0, 100, 110, 120, 130Gy were performed to measure absorbed dose and colony formation or not. BM03 (1×10.sup.7 cells/ml/vial) cryopreserved after filling in a vial was irradiated using a high-level irradiator at the Advanced Radiation Technology Institute of Korea Atomic Energy Research
[0207] Institute, and absorbed dose in each test was measured using an Alanine dosimeter. The results were confirmed as shown in Table 12 below.
TABLE-US-00012 TABLE 12 High level Primary experiment Secondary experiment Irradiation Absorbed Colony Absorbed Colony dose dose Formation Suitable/ dose Formation Suitable/ (Gy) (Gy) or not unsuitable (Gy) or not unsuitable 0 Gy — — — — — — 30 Gy 31.8 ◯ unsuitable — — — 40 Gy 40.4 ◯ unsuitable — — — 50 Gy 52.2 ◯ unsuitable — — — 60 Gy 61.2 ◯ unsuitable — — — 100 Gy — — — 100 X suitable 110 Gy — — — 110 X suitable 120 Gy — — — 120 X suitable 130 Gy — — — 130 X suitable
Experimental Example 9.1. Colony Formation or Not of the Irradiated BM03 Cell Line
[0208] In order to confirm whether colonies were formed on the BM03 cells of Experimental Example 9, it was confirmed whether colony formation of the cell line was made in the same manner as in Experimental Example 7.1. In Experimental Example 9, as a result of confirming whether colonies were formed in BM03 irradiated with low-level gamma rays and BM03 irradiated with high-level gamma rays, as shown in Tables 11 and 12 above, when irradiated with low-level gamma rays, colonies were formed, especially at an irradiation dose of 50 Gy or less, and when irradiation with high-level gamma rays, colonies were formed at an irradiation dose of 60 Gy or less, confirming that it was not suitable for use as a clinical sample. However, it was confirmed that colonies were not formed at 70 Gy or more for low-level gamma rays and 100 Gy or more for high-level gamma rays.
Experimental Example 9.2. Cell Number Measurement of the Irradiated BM03 Cell Line
[0209] With respect to the cells cultured in Experimental Example 9.1, in the case of the BM03 cell line irradiated with low-level gamma rays, on days 2, 7, 14, 28, 35, 42, and 49 from the cell thawing day, it was confirmed whether the number of cultured cells increased by counting the number of the attached cells.
[0210] As a result, as shown in Table 13 below and
TABLE-US-00013 TABLE 13 Dose Time Item 0 Gy 10 Gy 20 Gy 30 Gy 40 Gy 50 Gy 60 Gy 70 Gy Day 0 Number of viable 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 (inocula- cells (× 10.sup.5) tion) Viability (%) 89.9 89.1 93.6 92.0 93.6 92.0 90.8 90.6 Day 2 Number of viable 8.0 3.2 2.6 2.6 2.8 2.6 2.6 2.4 cells (× 10.sup.5) Viability (%) 84.3 70.0 52.5 60.8 55.5 56.6 47.2 49.8 Day 7 Number of viable 14.3 6.5 2.8 2.0 2.2 1.9 1.8 2.1 cells (× 10.sup.5) Viability (%) 93.1 91.2 75.8 79.8 81.7 86.2 86.3 78.0 Day 14 Number of viable 21.6 18.4 12.3 4.4 2.1 1.1 1.1 1.1 cells (× 10.sup.5) Viability (%) 92.0 94.4 90.5 78.6 73.8 59.2 63.2 70.8 Day 21 Number of viable 10.4 18.4 17.6 14.6 9.1 1.9 1.1 0.7 cells (× 10.sup.5) Viability (%) 86.7 91.8 92.9 86.1 86.9 67.4 62.3 69.8 Day 28 Number of viable — — — 17.3 8.1 2.3 0.4 0.6 cells (× 10.sup.5) Viability (%) — — — 93.8 86.0 88.7 74.6 73.0 Day 35 Number of viable — — — — — 6.0 0.3 0.3 cells (× 10.sup.5) Viability (%) — — — — — 79.8 61.3 47.9 Day 42 Number of viable — — — — — 3.0 2.1 0.1 cells (× 10.sup.5) Viability (%) — — — — — 35.4 66.9 68.1 Day 49 Number of viable — — — — — 6.0 2.7 0.1 cells (× 10.sup.5) Viability (%) — — — — — 68.9 49.3 16.3
[0211] In addition, for the BM03 cell line irradiated with high-level gamma rays in the same manner, the number of attached cells was counted on days 7, 14, 21, 28 (primary), and 7, 14, 21, 28, 35, 42, 49, 63 (secondary) from the day of cell thawing.
[0212] As a result, as shown in Table 14 below and
[0213] From these results, it can be seen that in the case of the BM03 cell line, the minimum absorbed dose should be 60 Gy or more.
TABLE-US-00014 TABLE 14 Dose (secondary experiment) Dose (primary experiment) 100 110 120 130 TIME ITEM 0 Gy 30 Gy 40 Gy 50 Gy 60 Gy 0 Gy Gy Gy Gy Gy Day 0 Number of 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 cells (× 10.sup.5) Viability 84.23 86.18 86.58 87.33 92.52 82.0 72.5 85.4 84.5 85.2 (%) Day 7 Number of 25.08 1.43 1.08 1.01 1.42 9.69 0.98 0.98 0.80 0.93 cells (× 10.sup.5) Viability 93.75 83.64 80.17 85.41 97.92 90.9 71.8 66.1 75.7 77.1 (%) Day 14 Number of 14.92 15.17 6.91 1.97 0.83 11.50 0.76 0.80 0.84 0.96 cells (× 10.sup.5) Viability 89.8 88.0 88.3 79.8 67.0 93.7 72.5 77.8 68.9 71.0 (%) Day 21 Number of 10.58 15.17 11.08 11.42 3.50 — 0.78 0.62 0.61 0.58 cells (× 10.sup.5) Viability 88.2 90.6 85.7 87.0 83.9 — 77.8 77.4 74.1 67.1 (%) Day 28 Number of — — — 12.67 10.50 — 0.42 0.46 0.37 0.32 cells (× 10.sup.5) Viability — — — 95.0 90.0 — 57.2 69.0 56.7 68.1 (%) Day 35 Number of — 0.39 0.23 0.27 0.28 cells (× 10.sup.5) Viability — 79.7 61.0 68.0 58.3 (%) Day 42 Number of — 0.33 0.36 0.31 0.25 cells (× 10.sup.5) Viability — 63.7 71.4 79.7 66.8 (%) Day 49 Number of — 0.25 0.15 0.23 0.16 cells (× 10.sup.5) Viability — 59.6 61.8 50.5 59.4 (%) Day 63 Number of — 0.08 0.07 0.09 0.13 cells (× 10.sup.5) Viability — 44.3 41.7 60.5 62.3 (%)
Experimental Example 9.3. Confirmation of Gene Expression Level in the Irradiated BM03 Cell Line
[0214] In order to measure the change in the cell titer after irradiation, after selecting a primer capable of confirming the inserted therapeutic genes TRAIL and CD::UPRT, the expression of the inserted gene was quantitatively measured through RT-qPCR reaction.
[0215] After harvesting cells immediately after thawing, and harvesting cells cultured for 2 days, 7 days and 14 days, respectively, RNA was extracted using Trizol, and cDNA was synthesized from 1 ug of RNA using 5× Prime Script RT Master Mix (Takara), and then RT-qPCR was performed using primers capable of specifically binding to each of TRAIL and CD.
[0216] As a result, as shown in
Experimental Example 10. In Vitro Confirmation of Tumorigenicity of BM102 Cell Line
[0217] The presence or absence of colony formation by anchorage-independent growth was confirmed in bone marrow-derived MSC, BM102 cell line, positive control (HeLa) and negative control (NIH3T3) in Soft Agar Gels using CytoSelect™ 96-well Cell Transformation Assay, and the presence or absence of tumor formation by cell transformation was quantified by measuring the absorbance by staining with MTS solution.
[0218] As a result, as shown in
Experimental Example 11. In Vitro Confirmation of Nerve Cell Protective and Therapeutic Effects of BM102 Cell Line
[0219] In order to confirm the nerve cell protective and therapeutic effects of the BM102 cell line, after co-culturing C6 glial cells, known to grow well by BDNF, with the BM102 cell line, an increase in the proliferation rate of C6 cells was confirmed in a cell number-dependent manner.
[0220] Specifically, C6 cells and BM102 cell line were co-cultured using a trans-well. After inoculating the same 2'10.sup.3 C6 cells in a 96-well plate, the BM102 cell line was diluted by ½ from 1×10.sup.4 cells and was inoculated into the trans-well to vary the number of co-cultured cells. After 24 hours of co-culture, the proliferation rate of C6 cells by the BDNF protein expressed in the BM102 cell line was quantified by treating the C6 cell medium in culture with MTS solution and measuring the absorbance.
[0221] As a result, as shown in
[0222] From the above results, it was confirmed that the BM102 cell line can stably mass-produce BDNF enough to be used as a cell therapeutic agent, and that the BM102 cell line stably expressing BDNF can be used as a cell therapeutic agent for protecting or treating nerve cells.
[0223] Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology
[0224] Accession Number: KCTC13876BP
[0225] Date of deposit: Jul. 2, 2019