BHC006-O2 Cell Line Screening Research Report

BHC006-O2 Cell Line Screening Study Report

Research purpose:

This research plan is based on BHC006 cells (HEK293T cells), which will be transfected with the BH504 plasmid (containing the gp gene and puromycin resistance), and selected using puromycin to obtain the BHC006(BH504) cell pool. Then, the BH506 plasmid (containing the envelope gene and blasticidin resistance) will be transfected into the BHC006(BH504) cell pool, selected using blasticidin, and double-positive cells will be screened out to form a stable expression of the retroviral gp protein and envelope protein in the BHC006-O2 cell poolNext, the method of single-clone screening was used to select high-yield cells from the BHC006-O2 cell pool, obtaining the BHC006-O2 cell line.

This study was based on the BHC006 cells (HEK293T cells). The BH504 plasmid (containing the gp gene and puromycin resistance) was transfected into BHC006 cells, and puromycin was used for selection to obtain the BHC006(BH504) cell pool. Then, the BH506 plasmid (containing the envelope gene and blasticidin resistance) was transfected into the BHC006(BH504) cell pool, and blasticidin was used for selection. Double-positive cells were screened out to form a stable BHC006-O2 cell pool expressing retroviral gp and envelope proteins. Subsequently, a monoclonal selection method was used to isolate high-producing cells from the BHC006-O2 cell pool to obtain the BHC006-O2 cell line..

Responsibilities and division of labor:

Research basis:

Ward, M. A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells. Mol. Ther. (2003) doi:10.1016/S1525-0016(03)00263-6.

S. Coroadinha, A. et al. Production of Retroviral Vectors: Review. Curr. Gene Ther. 10, 456–473 (2010).

Ghani K, Boivin-Welch M, Roy S, Dakiw-Piaceski A, Barbier M, Pope E, Germain L, Caruso M. Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells. Mol Ther Methods Clin Dev. 2019 Jun 7;14:90-99.

Experimental supplies:

Experimental sample

Experimental instruments and equipment

Experimental reagents and consumables

Experimental content:

Experiment Overview

This experiment is based on BHC006 cells, where linearized BH504 plasmid and linearized BH506 plasmid were sequentially transfected into BHC006 cells. After pressure screening and selecting double-positive cells, several clones were obtained through monoclonalization. The RV-GFP plasmid was transfected into monoclonal cells, and the monoclonal cells with the highest GFP titer were selected to establish the BHC006-O2 stable cell line.

The expected quality objectives of this experiment are as follows:

Table 1. List of expected quality objectives for this experiment

Cell doubling time DT=t* [lg2/ (lgNt-lgN0)]

*Doubling Time DT* = t* [lg2/ (lgNt-lgN0)]

Experimental design

The design of this study is as follows:

Experimental results

B HC006 Cell Preparation (PD-CTD402-01801, Experiment Start and End Time: 2022.04.23-2022.04.28)

 
Refer to "SOP-RD-307.00 Cell Culture Operation Procedures" for BHC006 cell resuscitation and expansion, prepare ≥ 1 × 10^6 cells. Cell number: 20426CE31, viability: 96.54%, density: 1.84E+06 cells/ml.

Refer to the 'SOP-RD-307.00_Cell Culture Operating Procedure' for the recovery and expansion of BHC006 cells, preparing ≥ 1×10⁶ cells. Cell ID: 220426CE31, viability: 96.54%, density: 1.84E+06 cells/ml

B HC006(BH504) cell pool preparation (PD-CTD402-01802, experiment start and end time: 2022.04.29-2022.06.01)

The day before transfection, the B HC006 cells were expanded to 1.0 E+0.5/cm² in a six-well plate. On the day of the experiment, B H504 plasmid transfection was performed, with a plasmid amount of 2 μg/well and using HP transfection reagent at a dosage of 6 μg/well.After transfection for 2 to 4 hours, the cells were expanded to a 6-well plate. The next day, 2 μg/ml, 5 μg/ml, 10 μg/ml, 12 μg/ml, 15 μg/ml, and 20 μg/ml of puromycin were added for selection. After 3-10 days, cells resistant to 2 μg/ml, 10 μg/ml, and 20 μg/ml puromycin were selected for G/P gene copy number detection. Three cell pools were used for GGene integration was successful, selecting a suitable copy number for the cell pool (20 μg/ml, GP gene copy number 2 4.5 copies/cell), and further inserting the R D114 gene.

On the day before transfection, BHC006 cells were expanded to 1.0E+05 cells/cm² in a 6-well plate. On day0 of transfection, the cells were transfected with 2 μg of BH504 plasmid per well, using 6 μl of HP transfection reagent per well. After 24 ± 4 hours post-transfection, the cells were expanded to six wells of a 6-well plate. The next day, puromycin was added at concentrations of 2 μg/ml, 5 μg/ml, 10 μg/ml, 12 μg/ml, 15 μg/ml, and 20 μg/ml for selection. After 3–10 days, puromycin-resistant cells at 2 μg/ml, 10 μg/ml, and 20 μg/ml were selected for GP gene copy number detection. Three cell pools showed successful integration of the GP gene. The cell pool with an appropriate copy number (20 μg/ml, GP gene copy number 24.5 copies/cell) was selected for further RD114 gene insertion..

Table 2. B HC006(BH504) cell pool GP gene copy number detection results

B HC006-O2 cell pool preparation (PD-CTD402-01803, experiment start and end time: 2022.06.02-2022.10.20)

 
Attempts were made to use HP transfection reagent and electroporation transfection method for the insertion of BH506 plasmid. First, the insertion of BH506 plasmid was attempted using HP transfection reagent, but after three rounds of HP transfection, the gene copy number of BH506 remained low. Then, the electroporation method was tried for transfection, ultimately obtaining a cell pool of BHC006-O2 containing a higher amount of BH506 plasmid genes.

HP transfection reagent and electroporation methods were separately tested for the insertion of the BH506 plasmid. First, HP transfection reagent was used to insert the BH506 plasmid. After three rounds of transfection, the BH506 gene copy number remained low. Electroporation was then attempted for transfection, resulting in a BHC006-O2 cell pool with a higher BH506 plasmid gene copy number.

HP 法 B H506 transfection

 
HP transfection method B H506 is the same as method 6.2. After the first transfection of B H506, a selection with about 3 weeks of puromycin was performed, resulting in the acquisition of the B HC006-O2 cell pool. Cells were taken for copy number detection of the G P gene and R D114 gene, with the results shown in Table 3. Due to the low copy number of R D114, a second and third HP transfection were subsequently performed. After 1 to 3 weeks of pressure selection, detection of the copy numbers of G P and R D114 genes was conducted. The results are shown in Table 3.After three transfections, the gene copy number of R D114 is still relatively low, with no significant increase. Therefore, we consider using electroporation for transfection.

The HP transfection method for BH506 plasmid insertion was the same as described in section 6.2. After the first BH506 transfection, the cells were subjected to blasticidin selection for approximately 3 weeks, resulting in the BHC006-O2 cell pool. The cells were then analyzed for GP and RD114 gene copy numbers, with the results shown in Table 3. Due to the low RD114 copy number, a second and third round of HP transfection was performed. After 1 to 3 weeks of selective pressure, GP and RD114 gene copy numbers were again analyzed, with results shown in Table 3. Despite three rounds of transfection, the RD114 gene copy number remained low with no significant increase. Therefore, electroporation was considered as an alternative method for transfection..

Table 3. HP Transfection B HC006-O2 Gene Copy Number Detection

Electroporation Method B H506 Transfection

 
The BHC006 (BH504) cell pool was transfected with BH506 using electroporation. The transfection culture dish was a six-well plate, with a cell amount of 1.23 M and a plasmid amount of 4 μg; the electroporation transfection buffer: DPBS; electroporation transfection parameters: voltage 110V, time 25 ms, pulse number 1 time, electrode plate spacing 2.0 mm.After transfection, the cells in the 6-well plate were cultured for recovery. Once the cell condition returned to normal, 15 μg/ml of puromycin was used for selection. After 8 days of selection, a puromycin-resistant B HC006-O2 cell pool was obtained. After continuing culture for 2 weeks, the B HC006-O2 cell pool was taken to detect the copy number of the G, P, and R D114 genes. The detection results are shown in Table 4.It can be seen that the R D114 gene has been successfully integrated into the B HC006 (B H504) cells, but the G P gene copy number level is lower than that of the B HC006 (B H504) cell pool, which may be due to detection errors, because the G P gene copy number level is close to that of R D114. Subsequent high copy number cell lines can be obtained through screening, so the next step of the experiment will continue.

Table 4. Electroporation method transfection B HC006-O2 gene copy number detection

B HC006-O2 Positive Monoclonal Screening (PD-CTD402-0180 4, Experiment Start and End Time: 2022.12.20 - 2023.04.10)

The dual-resistant cells obtained in section 6.3.2 were cultured in D10 complete medium containing 20μg/ml puromycin and 15μg/ml blasticidin. After the cell status recovered, subcloning was performed with a plating density of 0.8 cells/well. A total of 100 96-well plates (plate number YL221220CE10-1~100) were set up. Cloneselect Imager was used for photography to record the cell status.Cell culture 2 to 3 weeks later, perform cell amplification and toxin identification on the wells where cell growth appears, cell amplification records are in attachment A-08.

Toxin identification

Using RV-GFP plasmid transient amplification of monoclonal cells, detect 48h or 72h virus stock supernatant, and mark clones with CAR+ rate ≥ 5% as positive. This batch detected a total of 18 positive cell lines, and the test results are shown in Table 4 and Table 5. Flow cytometry results are in Attachment A-09.

Table 4. Toxicity Detection Statistics

Table 5. Toxicity Detection (CAR+ Rate)

Original data save path: Z:\Department files\R&D Department\R&D Department common files\Material information summary - All experimental personnel\Instrument equipment data\bdfaces\puzhu\puzhu\puzhu\nalm6\230119-GFP_5~8; Z:\Department files\R&D Department\R&D Department common files\Material information summary - All experimental personnel\Instrument equipment data\bdfaces\puzhu\puzhu\puzhu\nalm6\230119-GFP_13~15 51

G P, R D114 mRNA copy number detection

To determine the expression of positive cell-related genes at the mRNA level, 15 strains of cells that tested positive for toxin production were sent for testing of G, P, R, D114 mRNA copy numbers. Please refer to test report number T2023012801, and the test results can be found in Attachment A-07.

Table 6. BHC006-O2 Clone mRNA Copy Number Detection

Note: Among them, the monoclonal cells 2 3D12, 41D11, 18D8, 22C6, 2 2C4, 23G3, 23D11, 23E4, 4 4F2, 4 8C9, 2 5B3, 2 5D1, 1 3E5, 2 3C2, 4 5F2 are non-monoclonal cells, and subsequent research will prioritize monoclonal cells.

Toxin production confirmation

Instant transferRV-GFPToxin production confirmation of plasmid

The toxin production confirmation was conducted using 6 cell lines with higher copy numbers selected from section 6.4.3. A total of 1E+06 cells per well were plated in a 6-well plate, and the next day, RV-GFP plasmid transfection was performed. Viruses were collected for titer detection 72 to 96 hours later. The results of the toxin production confirmation are shown in Figure 2. The results indicate that all 6 cell lines can produce toxins, confirming they are positive cells. Among them, 22C6 and 22C4 had a higher virus transduction positive rate, followed by 23D12 and 23E4. Further comparisons of virus packaging effects will continue.

Figure 2. BHC006-O2 Instant Transfer RV-GFP Plasmid Toxin Production Confirmation

Original data save path: Z:\Department Files\Plasmid Virus Process Production Department\Shared Folder\11. FACSCelesta\zhilibingdu\titer-nalm6\20230219 BHC006-O2 GFP

Instant transfer BH P015 Plasmid toxin production confirmation

To verify the expression effect of the target plasmid, the BHP015 plasmid was transiently transfected into the above four single clone cell lines: 22C4, 22C6, 23D12, and 23E4, for toxin production identification. The experimental method can be referred to in section 6.4.3.1. Transfection culture bottle: T25, with reagent amounts scaled up proportionally. According to the titer detection results, all four cell lines can package BHV015 virus, with the highest titer being 22C6 clone - 96h virus, 7.25E+04 TU/ml. The transient virus titer is relatively low, and the next step will attempt to use a more efficient viral transduction method for toxin production detection.

Figure 3. BHC006-O2 Instant Transfer BHP015 Plasmid Toxin Confirmation

Original data save path: Z:\Department files\Plasmid virus process production department\Shared folder\11. FACSCelesta\zhilibingdu\titer-nalm6\20230301 BHC006-O2 +BHP015

Transduction BHV015B Virus toxin production confirmation

Using B HV015B concentrated toxin transduction 2 2C4, 2 2C6, 2 3D12, 2 3E4 four strains of cells, after the cells grow to a certain number, conduct toxin production identification. Toxin production conditions: 32℃, 5% CO₂, collect virus for titer determination after 72h. The results show that all four strains of cells can produce toxins, and the virus titer of 2 2C6 is the highest, with an infectious titer of > 1.32E+06 TU/ml.

Figure 4. BHC006-O2 Transduction BHV015B Virus Toxin Production Confirmation

Original data save path: Z:\Department Files\Plasmid Virus Process Production Department\Shared Folder\11. FACSCelesta\zhilibingdu\titer-nalm6\20230307 BHC006-O2 + BHV015B

Transduction BHV021-VSVG Virus production confirmation

To verify the packaging effects of 22C6 and 22C4 in the TA310 project, the BHV021-VSVG virus concentrated solution was used to transduce 22C6 and 22C4 cells for virus production testing. Production conditions: 32°C, 5% CO₂, collect virus at 72h and 96h for titer determination. The results showed that both cell lines could produce virus, with 22C6 being slightly better, consistent with the results in 6.4.3.3, further validating the conclusion that the monoclonal strains 22C6 and 22C4 can be used for virus packaging.The cells with a higher toxin production titer of 2C6 are named HC006-O2 and are stored for future use after freezing.

Figure 5. BHC006-O2 Transduction BHV021-VSVG Virus Titer Confirmation

Original data save path: Z:\Department Files\Plasmid Virus Process Production Department\Shared Folder\10. FACSCelesta\zhilibingdu\titer-nalm6\20230406 BHC006-O2-BHV021 TITER

Cell cryopreservation

Selection of toxin detection titers with the highest being 15 strains of cells (23D12, 44F2, 48C9, 25B3, 25D1, 41D11, 13E5, 18D8, 22C6, 22C4, 23G3, 23C2, 45F2, 23D11, 23E4) for cryopreservation, using a cryopreservation solution of 90% D10 culture medium + 10% DMSO. Each of the 15 strains of cells is cryopreserved in 3 vials, 1ml per vial.

B HC006-O2 Monoclonal Traceability

On the first day after subcloning, use Clone Select Imager to take photos and record cell status, continuously recording cell status. Figure 6 shows photos from the first day after subcloning, the sixth day, and the last day before cell expansion. It can be seen that BHC006-O2 is derived from a single cell.

Figure 6. BHC006-O2 Cell Image

BHC006-O2-PCBOriginal Cell Bank andBHC006-O2-RCBResearch Cell BankLibrary Construction

Revive a22C6（BHC006-O2）cells,expanded to5T175, after trypsin digestion, using cryopreservation solution（90%D10medium+10% DMSO）resuspended,with1ml/tubepackaged,10tubesasPCBoriginal cell bank,20tubesasRCBresearch cell bank.

Table 7. BHC006-O2 Library Information

After freezing, take 1 sample of RCB bank cells to test the recovery rate, the cell recovery rate is 93.53% > 80%, meeting the library construction requirements. Take 3 samples of B HC006-O2-RCB cells for testing for sterility, mycoplasma, and endotoxin, the results all meet the library construction requirements, please refer to the test number: T2023032702, feedback results see attachment A-16.

B HC006-O2 Growth Curve Determination

B HC006-O2 inoculated at 4E+04 cells/cm² in 6-well plates, every 24 h ± 2 h select one well for trypsin digestion, resuspend in 1ml D10 complete medium, and count. Using culture time (h) as the x-axis and total cell density (E+06 cells) as the y-axis, plot the cell growth curve. From the growth curve, it can be seen that B HC006-O2 has a logarithmic growth phase from 24 h to ~96 h post-inoculation; the doubling time during the logarithmic growth phase is 24.13h＜48h, meet the expected quality target.

Experimental conclusion:

This study transfected linearized BH504 plasmid and linearized BH506 plasmid into BHC006 cells through chemical transfection and electroporation, obtaining a cell pool for virus packaging. Based on this, monoclonal selection was performed to obtain a monoclonal cell line named BHC006-O2.

Cell growth curve shows that BHC006-O2 has a logarithmic phase doubling time of 24.13 h, which meets the expected quality target.

Virus transduction test shows that BHC006-O2 cells can be used as platform cells for subsequent screening of stable retroviral production strains.

The corresponding cells have been established, PCB and RCB, and the test results meet the expected quality standards and library construction standards. They can be used for the development of toxin-producing cell lines in subsequent projects.

Attachment:

"BHC006-O2 Cell Line Screening Study - BHC006 Cell Preparation Experimental Record" (PD-CTD402-018-01, total 10 pages)

"BHC006-O2 Cell Line Screening Study - BHC006 (BH504) Cell Pool Preparation Experimental Record (PD-CTD402-018-02, Total 27 Pages)"

"BHC006-O2 Cell Line Screening Study - BHC006 - O2 Cell Pool Preparation Experimental Record" (PD-CTD402-018-03, total 70 pages)

"BHC006-O2 Cell Line Screening Study - BHC006-O2 Positive Monoclonal Screening Experiment Record" (PD-CTD402-018-04, total 84 pages)

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