As the market invests in allogeneic cell and gene therapies, large-scale expansion of stem cells has gained significant interest (1). To ensure low volume at the end of the process, volume reduction is a crucial factor in evolving workflows. To meet these needs, we propose using the Sepax™ C-Pro cell-processing system to automate the workflow, while ensuring key cell properties including high viability, recovery, and pluripotency. In this study, we implemented the CultureWash C-Pro software to meet these requirements in a functionally closed system. By applying insights from our previous studies, we created two novel parameter sets to process bone marrow-derived mesenchymal (MSCs) and induced pluripotent (iPSCs) stem cells. We demonstrate a protocol that achieved > 82% recovery without a negative impact on cells' viability or pluripotency.
Introduction
Due to the intricacy of stem cell culture, both in 2D monoculture and 3D spheroid, we sought to develop a parameter and reagent set that both limited cell loss during concentration steps and prevented cells from sticking to the disposable kits. Utilizing lessons learned from our previous study Tuning intermediate volumes on Sepax™ C-Pro cell processing system, we tested harvest recoveries for MSCs and iPSCs to illustrate volume reduction and buffer exchange capabilities in a functionally closed system. Overall, the study aimed to:
- Confirm the proposed intermediate volume (IV) based on cell numbers for new cell types of different diameters.
- Evaluate recovery of 2D and 3D cultures using CultureWash C-Pro software.
- Verify recovery, viability, and pluripotency are maintained during processing steps.
Based on our prior studies, we implemented the following changes: 1. adjusted filling/extraction speeds of the cellular product in the processing chamber to avoid cell loss, 2. enabled the syringe-based manual extraction (ME) parameter to accommodate low final volumes while minimizing cell loss, 3. ensured a large enough intermediate volume (IV) to contain the total cell number, based on the cells’ specific diameter.
Additionally, we used the CT-90.1 Sepax™ C-Pro kit, designed for final volumes of 20 mL or less, as this kit has additional pathways specifically designed for the ME mode.
Materials and methods
Cell culture
MSC
Bone-marrow-derived MSC were expanded from a 1 mL vial of ~ 1 × 106 cells frozen in a cryoprotectant. Cells were resuspended in ATCC basal medium for MSC, supplemented with ATCC MSC supplement kit and penicillin/streptomycin (100×, Hyclone™ serum), and seeded in a T-175 flask. A full media exchange was done 24 h after seeding and every 2 d thereafter. Cells were cultured until they reached ~ 80% confluence and then washed twice with HyClone™ Dulbecco’s phosphate buffered saline (DPBS) and incubated with 4 mL of 0.25% Trypsin solution for 5 min, at 37°C. Flasks were then washed with 8 mL of the complete media composition mentioned above and centrifuged at 300 × g for 5 min. After resuspending the pellet in completed media, the cells were split into three new flasks. This passaging procedure was performed in a similar way before processing with Sepax™ C-Pro cell processing system, but cells were resuspended in a volume of complete media between 20 and 40 mL.
iPSC
Bone-marrow derived induced pluripotent stem cells (iPSCs) were expanded on cell basement membrane-coated T-flasks using feeder-free medium. Medium was supplemented with 10 µM of Rho-kinase inhibitor for 24 h post passage. Cells were cultured until they reached ~ 80% confluency with daily media exchange. iPSCs were rinsed with HyClone™ Dulbecco’s Modified Eagle Medium (DMEM) F:12, dissociated using 0.5 U/mL of stem cell dissociation agent, and centrifuged at 300 × g for 10 min. iPSCs were split using a 1:5 ratio into new precoated T-175 flasks for expansion.
For further expansion, a mini bioreactor system was implemented, using single-use Eppendorf® BioBLU® 0.3c vessels. iPSCs were seeded at a density of 0.25 million cells/mL at a working volume of 160 mL of feeder-free medium supplemented with 10 µM Rho-kinase inhibitor. Conditions included 40% dissolved oxygen (DO) control, pH 7.2, at 37°C and 189 rpm. Perfusion was implemented starting at day 1, ramping up from 0.5 to 2× perfusion over 6 to 7 d. iPSC aggregates were then harvested ranging in volumes between 180 and 210 mL.
Sepax™ C-Pro system processing
After the cells were transferred to the initial bag for processing on the Sepax™ C-Pro system, cell counts were taken on the NucleoCounter® NC-200 cell counter (ChemoMetec A/S). The initial volume was kept below 220 mL (equivalent to the capacity of the processing chamber) to ensure all replicates had the same number of concentration cycles (1 cycle), to reduce variability. The wash solution was PBS/EDTA buffer supplemented with 0.5% of human serum albumin. Parameters selected on CultureWash C-Pro software were as mentioned in Table 1. We executed all the on-screen application prompts, including the manual extraction steps requiring a syringe and manual extraction tool.
Table 1. CultureWash C-Pro software parameters
| Sepax™ C-Pro system parameters |
Units |
Parameters |
|
|---|---|---|---|
| MSC |
iPSC |
||
| Initial volume |
mL | 20–41 |
180–210 |
| Detect initial volume |
No (0) Yes (1) |
0 | |
| Dilution ratio | 0 | ||
| Dilution speed |
mL/min |
NA | |
| 60 s mixing after dilution |
No (0) Yes (1) |
NA |
|
| Thaw bag validation |
No (0) Yes (1) |
1 | |
| Hang bag validation |
No (0) Yes (1) |
1 | |
| Input bag rinsing |
No (0) Yes (1) |
1 | |
| Input bag rinse volume |
mL |
50 | |
| Pause input bag rinse |
No (0) Yes (1) |
1 | |
| Optical cell detection |
No (0) Yes (1) |
1 | |
| Product filing speed |
mL/min |
80 | |
| Waste extraction speed |
mL/min |
80 | |
| Intermediate volume |
mL |
10 | |
| Prewash cycles |
cycles |
1 | |
| Prewash g-force |
× g |
400 | |
| Prewash sedimentation time |
Seconds (s) |
300 | 420 |
| Switch washing solution |
No (0) Yes (1) |
0 | |
| Post-wash cycles |
cycles |
0 | |
| Post-wash g-force |
× g |
NA | |
| Post-wash sedimentation time |
s |
NA | |
| Switch resuspension solution |
No (0) Yes (1) |
0 | |
| Exchange waste bags |
No (0) Yes (1) |
0 | |
| Final volume |
mL |
20 | |
| Manual extraction |
No (0) Yes (1) |
1 | |
| Syringe for manual extraction | No (0) Yes (1) | 1 | |
Flow cytometry
MSC
CD73, CD90, and CD105 markers were quantitated to measure pluripotency using the MSC flow kit. 5 × 105 trypsinized cells were aliquoted into flow tubes and washed twice in DPBS (300 ×g, 10 min). 20 µL of the antibody cocktail was added and allowed to stain at 4°C, for 30 min. Cells were then washed, resuspended in flow buffer, and run on Beckman Coulter CytoFLEX® S platform and analyzed using FlowJo software.
iPSC
Pluripotency was quantitated using a pluripotent stem cell multicolor flow cytometry kit. 1 × 106 cells were aliquoted and washed with DPBS twice (300 ×g, 10 min). Rinsed iPSCs were fixed at 4°C, for 30 min), permeabilized, stained for 45 min, at ambient temperature, rinsed, and resuspended. Samples were measured using CytoFLEX® S platform and analyzed using FlowJo software.
Recovery
Recovery was defined as the ratio of total viable cell number (TVCN) in the final vessel over the TVCN in the initial vessel. Results were calculated as the average of three counts on the NucleoCounter® NC-200 cell counter. To stay within the counting boundaries of the cell counter, samples were kept between 5 × 105 cell/mL and 3 × 106 cells/mL by diluting in HyClone™ DPBS (the maximum dilution required was 10×). No counts fell below the minimum range, so no concentration steps were required.
Analytics
To ensure proper cell growth and culture conditions, we used a cell culture analyzer. The NucleoCounter® NC-200 cell counter was used for all cell counts to measure viability and total cell number, in triplicate, using the Via1-cassettes. Samples were diluted in HyClone™ DPBS, as considered appropriate, prior to cell count. Volumes of the initial bag and mixing final vessel were measured by graduations in pipettes.
Results and discussion
Cell recovery and viability
MSC
Fig 1. (A) Recovery percentage of total viable cells for each run ranging from 10 × 106 (runs 1-3) and 15 × 106 TVCN (run 4). (B) Comparison of cell viability pre- and post-CultureWash C-Pro per run. (C) Flow cytometry gating illustrating pluripotency markers CD73, CD90, and CD105.
Triplicate runs (runs 1-3) with an input of 1 × 107 viable cells resulted in an average recovery of 90.0% using the CultureWash C-Pro parameters set as indicated in Table 1. Post-process viability remained high (> 98%), comparable to the pre-processing viability (97% to 98%) (Fig 1). Final product stemness was measured to be greater than 98% by co-expression of CD73, CD90, and CD105. To determine if greater input of cell number affected recovery and viability, we performed a run (run 4) with an input of 1.5 × 107 viable cells. Results showed a recovery of 99.3% with post-process viability of 98.5%, indicating that recovery was not lowered by increasing input cell number.
iPSC
Fig 2. (A) Recovery percentage of total viable cells for each run. (B) Comparison of cell viability pre- and post-CultureWash C-Pro software. (C) Flow cytometry gating illustrating pluripotency markers OCT3/4, SSEA4, and SOX2.
In efforts to remove the ME step, the first iPSC run (run 1) did not enable ME mode. Results showed an overall increase in viability from 63.1% (pre-processing) to 78.4% (post-processing) (Fig 2B), likely due to the centrifugation step that helps removing non-viable cells. However, we found that recovery was low and below 80% (72.2%), thus ME mode was enabled for runs 2 to 4.
The second run with an input of cell number of 1.8 × 108 viable iPSCs cells once again showed an increase in viability from 65.6% to 78% accompanied by a higher recovery of 85.1% (Fig 2A). For the next runs, we increased the input of cell numbers to 3.4 × 108 and 7.3 × 108 cells. On average, recovery using ME remained high, with 83.7% recovery and 76.6% post-process viability, regardless of cell number input.
To determine if processing affected iPSC pluripotency, flow cytometry was used to quantitate the expression of established embryonic stem cell markers: SOX2 and OCT3/4. Results showed > 87% staining for SEA4/OCT3_4/SOX2, indicating that the process did not alter iPSC pluripotency (Fig 2C).
Conclusion
- We maintained cell viability throughout the process and were able to remove dead cells when processing the 3D iPSC aggregates.
- Pluripotency phenotype was also preserved, key for continued cell growth and good starting material for differentiation and exosome collection. The CultureWash C-Pro parameters in this study could be set to requirements for ME due to the small final volume (< 20 mL).
- Cell loss can be greatly reduced using the guidance from our previous app notes (available here) by tuning the intermediate volume (IV) and rinse volume.
- Future studies will aim to tackle new guidelines when low extraction volume is not required, aiming at a more automatized process from start to finish, while minimizing cell loss.
CY38277-11Oct23-AN
As the market invests in allogeneic cell and gene therapies, large-scale expansion of stem cells has gained significant interest (1). To ensure low volume at the end of the process, volume reduction is a crucial factor in evolving workflows. To meet these needs, we propose using the Sepax™ C-Pro cell-processing system to automate the workflow, while ensuring key cell properties including high viability, recovery, and pluripotency. In this study, we implemented the CultureWash C-Pro software to meet these requirements in a functionally closed system. By applying insights from our previous studies, we created two novel parameter sets to process bone marrow-derived mesenchymal (MSCs) and induced pluripotent (iPSCs) stem cells. We demonstrate a protocol that achieved > 82% recovery without a negative impact on cells' viability or pluripotency.
Introduction
Due to the intricacy of stem cell culture, both in 2D monoculture and 3D spheroid, we sought to develop a parameter and reagent set that both limited cell loss during concentration steps and prevented cells from sticking to the disposable kits. Utilizing lessons learned from our previous study Tuning intermediate volumes on Sepax™ C-Pro cell processing system, we tested harvest recoveries for MSCs and iPSCs to illustrate volume reduction and buffer exchange capabilities in a functionally closed system. Overall, the study aimed to:
- Confirm the proposed intermediate volume (IV) based on cell numbers for new cell types of different diameters.
- Evaluate recovery of 2D and 3D cultures using CultureWash C-Pro software.
- Verify recovery, viability, and pluripotency are maintained during processing steps.
Based on our prior studies, we implemented the following changes: 1. adjusted filling/extraction speeds of the cellular product in the processing chamber to avoid cell loss, 2. enabled the syringe-based manual extraction (ME) parameter to accommodate low final volumes while minimizing cell loss, 3. ensured a large enough intermediate volume (IV) to contain the total cell number, based on the cells’ specific diameter.
Additionally, we used the CT-90.1 Sepax™ C-Pro kit, designed for final volumes of 20 mL or less, as this kit has additional pathways specifically designed for the ME mode.
Materials and methods
Cell culture
MSC
Bone-marrow-derived MSC were expanded from a 1 mL vial of ~ 1 × 106 cells frozen in a cryoprotectant. Cells were resuspended in ATCC basal medium for MSC, supplemented with ATCC MSC supplement kit and penicillin/streptomycin (100×, Hyclone™ serum), and seeded in a T-175 flask. A full media exchange was done 24 h after seeding and every 2 d thereafter. Cells were cultured until they reached ~ 80% confluence and then washed twice with HyClone™ Dulbecco’s phosphate buffered saline (DPBS) and incubated with 4 mL of 0.25% Trypsin solution for 5 min, at 37°C. Flasks were then washed with 8 mL of the complete media composition mentioned above and centrifuged at 300 × g for 5 min. After resuspending the pellet in completed media, the cells were split into three new flasks. This passaging procedure was performed in a similar way before processing with Sepax™ C-Pro cell processing system, but cells were resuspended in a volume of complete media between 20 and 40 mL.
iPSC
Bone-marrow derived induced pluripotent stem cells (iPSCs) were expanded on cell basement membrane-coated T-flasks using feeder-free medium. Medium was supplemented with 10 µM of Rho-kinase inhibitor for 24 h post passage. Cells were cultured until they reached ~ 80% confluency with daily media exchange. iPSCs were rinsed with HyClone™ Dulbecco’s Modified Eagle Medium (DMEM) F:12, dissociated using 0.5 U/mL of stem cell dissociation agent, and centrifuged at 300 × g for 10 min. iPSCs were split using a 1:5 ratio into new precoated T-175 flasks for expansion.
For further expansion, a mini bioreactor system was implemented, using single-use Eppendorf® BioBLU® 0.3c vessels. iPSCs were seeded at a density of 0.25 million cells/mL at a working volume of 160 mL of feeder-free medium supplemented with 10 µM Rho-kinase inhibitor. Conditions included 40% dissolved oxygen (DO) control, pH 7.2, at 37°C and 189 rpm. Perfusion was implemented starting at day 1, ramping up from 0.5 to 2× perfusion over 6 to 7 d. iPSC aggregates were then harvested ranging in volumes between 180 and 210 mL.
Sepax™ C-Pro system processing
After the cells were transferred to the initial bag for processing on the Sepax™ C-Pro system, cell counts were taken on the NucleoCounter® NC-200 cell counter (ChemoMetec A/S). The initial volume was kept below 220 mL (equivalent to the capacity of the processing chamber) to ensure all replicates had the same number of concentration cycles (1 cycle), to reduce variability. The wash solution was PBS/EDTA buffer supplemented with 0.5% of human serum albumin. Parameters selected on CultureWash C-Pro software were as mentioned in Table 1. We executed all the on-screen application prompts, including the manual extraction steps requiring a syringe and manual extraction tool.
Table 1. CultureWash C-Pro software parameters
| Sepax™ C-Pro system parameters |
Units |
Parameters |
|
|---|---|---|---|
| MSC |
iPSC |
||
| Initial volume |
mL | 20–41 |
180–210 |
| Detect initial volume |
No (0) Yes (1) |
0 | |
| Dilution ratio | 0 | ||
| Dilution speed |
mL/min |
NA | |
| 60 s mixing after dilution |
No (0) Yes (1) |
NA |
|
| Thaw bag validation |
No (0) Yes (1) |
1 | |
| Hang bag validation |
No (0) Yes (1) |
1 | |
| Input bag rinsing |
No (0) Yes (1) |
1 | |
| Input bag rinse volume |
mL |
50 | |
| Pause input bag rinse |
No (0) Yes (1) |
1 | |
| Optical cell detection |
No (0) Yes (1) |
1 | |
| Product filing speed |
mL/min |
80 | |
| Waste extraction speed |
mL/min |
80 | |
| Intermediate volume |
mL |
10 | |
| Prewash cycles |
cycles |
1 | |
| Prewash g-force |
× g |
400 | |
| Prewash sedimentation time |
Seconds (s) |
300 | 420 |
| Switch washing solution |
No (0) Yes (1) |
0 | |
| Post-wash cycles |
cycles |
0 | |
| Post-wash g-force |
× g |
NA | |
| Post-wash sedimentation time |
s |
NA | |
| Switch resuspension solution |
No (0) Yes (1) |
0 | |
| Exchange waste bags |
No (0) Yes (1) |
0 | |
| Final volume |
mL |
20 | |
| Manual extraction |
No (0) Yes (1) |
1 | |
| Syringe for manual extraction | No (0) Yes (1) | 1 | |
Flow cytometry
MSC
CD73, CD90, and CD105 markers were quantitated to measure pluripotency using the MSC flow kit. 5 × 105 trypsinized cells were aliquoted into flow tubes and washed twice in DPBS (300 ×g, 10 min). 20 µL of the antibody cocktail was added and allowed to stain at 4°C, for 30 min. Cells were then washed, resuspended in flow buffer, and run on Beckman Coulter CytoFLEX® S platform and analyzed using FlowJo software.
iPSC
Pluripotency was quantitated using a pluripotent stem cell multicolor flow cytometry kit. 1 × 106 cells were aliquoted and washed with DPBS twice (300 ×g, 10 min). Rinsed iPSCs were fixed at 4°C, for 30 min), permeabilized, stained for 45 min, at ambient temperature, rinsed, and resuspended. Samples were measured using CytoFLEX® S platform and analyzed using FlowJo software.
Recovery
Recovery was defined as the ratio of total viable cell number (TVCN) in the final vessel over the TVCN in the initial vessel. Results were calculated as the average of three counts on the NucleoCounter® NC-200 cell counter. To stay within the counting boundaries of the cell counter, samples were kept between 5 × 105 cell/mL and 3 × 106 cells/mL by diluting in HyClone™ DPBS (the maximum dilution required was 10×). No counts fell below the minimum range, so no concentration steps were required.
Analytics
To ensure proper cell growth and culture conditions, we used a cell culture analyzer. The NucleoCounter® NC-200 cell counter was used for all cell counts to measure viability and total cell number, in triplicate, using the Via1-cassettes. Samples were diluted in HyClone™ DPBS, as considered appropriate, prior to cell count. Volumes of the initial bag and mixing final vessel were measured by graduations in pipettes.
Results and discussion
Cell recovery and viability
MSC
Fig 1. (A) Recovery percentage of total viable cells for each run ranging from 10 × 106 (runs 1-3) and 15 × 106 TVCN (run 4). (B) Comparison of cell viability pre- and post-CultureWash C-Pro per run. (C) Flow cytometry gating illustrating pluripotency markers CD73, CD90, and CD105.
Triplicate runs (runs 1-3) with an input of 1 × 107 viable cells resulted in an average recovery of 90.0% using the CultureWash C-Pro parameters set as indicated in Table 1. Post-process viability remained high (> 98%), comparable to the pre-processing viability (97% to 98%) (Fig 1). Final product stemness was measured to be greater than 98% by co-expression of CD73, CD90, and CD105. To determine if greater input of cell number affected recovery and viability, we performed a run (run 4) with an input of 1.5 × 107 viable cells. Results showed a recovery of 99.3% with post-process viability of 98.5%, indicating that recovery was not lowered by increasing input cell number.
iPSC
Fig 2. (A) Recovery percentage of total viable cells for each run. (B) Comparison of cell viability pre- and post-CultureWash C-Pro software. (C) Flow cytometry gating illustrating pluripotency markers OCT3/4, SSEA4, and SOX2.
In efforts to remove the ME step, the first iPSC run (run 1) did not enable ME mode. Results showed an overall increase in viability from 63.1% (pre-processing) to 78.4% (post-processing) (Fig 2B), likely due to the centrifugation step that helps removing non-viable cells. However, we found that recovery was low and below 80% (72.2%), thus ME mode was enabled for runs 2 to 4.
The second run with an input of cell number of 1.8 × 108 viable iPSCs cells once again showed an increase in viability from 65.6% to 78% accompanied by a higher recovery of 85.1% (Fig 2A). For the next runs, we increased the input of cell numbers to 3.4 × 108 and 7.3 × 108 cells. On average, recovery using ME remained high, with 83.7% recovery and 76.6% post-process viability, regardless of cell number input.
To determine if processing affected iPSC pluripotency, flow cytometry was used to quantitate the expression of established embryonic stem cell markers: SOX2 and OCT3/4. Results showed > 87% staining for SEA4/OCT3_4/SOX2, indicating that the process did not alter iPSC pluripotency (Fig 2C).
Conclusion
- We maintained cell viability throughout the process and were able to remove dead cells when processing the 3D iPSC aggregates.
- Pluripotency phenotype was also preserved, key for continued cell growth and good starting material for differentiation and exosome collection. The CultureWash C-Pro parameters in this study could be set to requirements for ME due to the small final volume (< 20 mL).
- Cell loss can be greatly reduced using the guidance from our previous app notes (available here) by tuning the intermediate volume (IV) and rinse volume.
- Future studies will aim to tackle new guidelines when low extraction volume is not required, aiming at a more automatized process from start to finish, while minimizing cell loss.
CY38277-11Oct23-AN
- Hoang DM, Pham PT, Bach TQ et al. Stem cell-based therapy for human diseases. Signal Transduct Target Ther. 2022 Aug 6;7(1):272. doi: 10.1038/s41392-022-01134-4. PMID: 35933430; PMCID: PMC9357075.