Mastering iNK Cell Generation From PSC Spheroids: A Streamlined Protocol

A streamlined protocol for generating iNK cells from PSC spheroids Stem cell research The cell therapy field has grown exponentially, offering promising therapeutics for several diseases. However, significant obstacles must still be overcome, including the ability to obtain large yields of the appropriate cell type. Natural killer (NK) cells are a cytotoxic, innate, lymphoid immune cell of interest in cancer cell therapy research. These innate immune cells can kill malignant cells without the need for HLA matching. NK cell therapy clinical trials require ~5 x 106 to 1 x 108 NK cells per kilogram of body weight, which underscores the need for improved scalability of cell manufacturing. To address this issue, scientists are exploring pluripotent stem cells (PSCs) as an easy-to-scale source of allogeneic, PSC-derived NK cells, or invariant NK (iNK) cells. Current PSC-to-iNK cell differentiation protocols include an embryoid body formation step and/or co-culture with feeder cells, which are inconsistent and lack scalability, hampering the manufacturing of large quantities of functional iNK cells. Here, we have developed a protocol to differentiate PSCs to iNK cells, which features the 3D PSC expansion capabilities of Gibco™ CTS™ StemScale™ PSC Suspension Medium. In this protocol, iNK cells are generated by selective application of several protein/small molecule cocktails, and without the use of embryoid body formation, scaffolds, or exogenous feeder cells. This differentiation protocol is presented in three sections: 1) PSC-to-induced hematopoietic progenitor cell (iHPC) differentiation, 2) iHPC-to-iNK cell differentiation, and 3) iNK expansion. PSC culture and iHPC differentiation are performed under constant agitation via an orbital shake platform, followed by a static culture to promote maturation and expansion of iNK cells. Protocol | CTS StemScale PSC Suspension Medium Day –5: 3D PSC culture 1. For detailed instructions on PSC suspension culture, follow guidelines provided in the CTS StemScale PSC Suspension Medium user guide. Notes: • For best differentiation results, adapt PSCs for suspension culture with three consecutive passages in CTS StemScale medium prior to starting the differentiation protocol. • This protocol’s guidance is based on using 6-well plates. Other formats are referenced throughout where steps may differ. Preparing growth factors and side solutions Reconstitute all growth factors according to manufacturer’s recommendations. Store aliquots of appropriate working concentrations at –80°C. Schedule for PSC spheroid culture: Day –5 Day –4 Day –3 Day –2 Day –1 Day 0 Seed 3D PSC culture Half media change Half media change Half media change Half media change Passage Figure 1. Overview of the PSC-to-iNK cell differentiation protocol. Images created by BioRender.com. • It will be necessary to determine an appropriate stirring speed when using equipment other than what is listed in this protocol. Please see the CTS StemScale PSC Suspension Medium user guide for help with speed conversions. • Optimization of protein/small molecule concentrations, seeding densities, and culture durations are recommended as cell line variability occurs. Prior to initiating this protocol, PSCs should be cultured in suspension for at least three passages in CTS StemScale PSC Suspension Medium. PSC spheroids iHPC induction Hematopoietic progenitor cells iNK cell induction iNK cell differentiation iNK cell maturation and proliferation CTS StemScale medium CTS StemScale medium StemPro-34 medium CTS NK-Xpander Medium, MEM α, Human AB Serum Suspension (orbital shaker/bioreactor) Static culture Y–27632 BMP4 BMP4 FLt3 IL–15 IL–15 DNase I Y-27632 bFGF SCF IL–7 IL–7 DNase I VEGF TPO IL–3 FLt3 VEGF SCF FLt3 SCF SCF SCF D(–5) D(0) D(2) D(5) D(6) D(9) D(15) D(38) PSC iHPC iNK cell 8 days 29 days 2 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Day 0: Spheroid formation Schedule for iHPC differentiation: Day 0 Day 1 Day 2 Day 5 Day 7 Day 9 Passage of 3D culture Half media change Full media change Full media change Half media change Half media change Mesoderm induction: Mesoderm induction medium 1 Component Stock conc. Final conc. CTS StemScale basal medium 1X 1X CTS StemScale supplement 10X 1X Antibiotic-Antimycotic 100X 1X Y-27632 10 mM 10 µM DNase I 1 U/µL 0.5 U/mL SCF 100 µg/mL 50 ng/mL BMP4 100 µg/mL 40 ng/mL VEGF 100 µg/mL 50 ng/mL 1. Prepare mesoderm induction medium 1 fresh and allow it to warm to room temperature. 2. Transfer the entire volume of spheroids from each well into separate 15 or 45 mL conical tubes. Note: Healthy spheroids should be ~400 µm in diameter and appear rounded with well-defined borders. The culture time to reach the recommended spheroid size may differ by cell line; it may take ~4–6 days. 3. Centrifuge the spheroids at 200 x g at room temperature for 2 minutes and aspirate medium, being careful not to disturb the spheroid pellet. Alternatively, allow the spheroids to settle by gravity for approximately 5 minutes and aspirate the medium. 4. Resuspend the cell pellet in 25% Gibco™ CTS™ TrypLE™ Select Enzyme (dilute with Gibco™ CTS™ DPBS). Note: Recommended dissociation conditions differ by vessel type (Table 1). Table 1. Dissociation conditions based on vessel type. Dissociation considerations Volume 6-well plate 125 mL flask 100 mL bioreactor 25% CTS TrypLE Select Enzyme 1 mL per well 5 mL 15 mL (50% CTS TrypLE Select Enzyme) Time 5 minutes 10 minutes 10–15 minutes 5. Place the conical tubes at 37°C in a water bath for 5 minutes. Note: Agitate the tube every few minutes to mix the spheroids in CTS TrypLE Select Enzyme to facilitate dissociation. 6. Triturate the cell suspension gently 2–3 times with a P1000 pipette to ensure spheroids are dissociated into single cells, and add 3 mL of Gibco™ DMEM/F12 + Gibco™ GlutaMAX™ Supplement per 1 mL of diluted CTS TrypLE Select Enzyme to the cell suspension. 7. Centrifuge the cells at 200 x g for 4 minutes at room temperature and aspirate supernatant, being certain not to disturb the cell pellet. 8. Resuspend the cell pellet in mesoderm induction medium 1 and perform assessment of total cell count and viability. Note: Resuspension volume varies by vessel type (Table 2). Table 2. Resuspension volume of mesoderm induction medium 1, based on vessel type. Working volume for different vessels Vessel Non–tissue culture treated 6-well plate Non–tissue culture treated 125 mL flask Non–tissue culture treated 100 mL bioreactor Volume 2 mL 5 mL 10 mL 9. Seed single cell suspension into a non–tissue culture treated vessel (6-well plate–100 mL bioreactor) at a final concentration of 3 x 105 cells/mL. To support proper spheroid formation in different vessels, add mesoderm induction medium 1 to a final volume as listed in Table 3. Note: Seeding density of starting PSCs may vary depending on cell line. Make sure to optimize this step for your cell line of interest. We recommend 3 x 105 cells/mL as a starting point for day 0. Table 3. Volume of mesoderm induction medium 1, based on vessel type. Working volume for different vessels Vessel Non–tissue culture treated 6-well plate Non–tissue culture treated 125 mL flask Non–tissue culture treated 100 mL bioreactor Volume 2 mL 20 mL 100 mL 10. Incubate plates at 37°C, 5% CO2 on an orbital shaker platform set to 70 RPM. Note: The recommended stir speed for a 100 mL vertical wheel bioreactor is 30–40 RPM but may require additional optimization. 3 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Figure 2. Morphological observations during iHPC differentiation. (A) Day 2: PSC spheroids aggregate and grow to ~150–250 µm diameter. At this stage, the individual cells that did not aggregate into spheroids are lost during subsequent medium changes. (B) Day 8: Increased iHPC population detaches from spheroids to further mature and expand in suspension. Day 1: Medium change Mesoderm induction: Mesoderm induction medium 2 Component Stock conc. Final conc. CTS StemScale basal medium 1X 1X CTS StemScale supplement 10X 1X Antibiotic-Antimycotic 100X 1X SCF 100 µg/mL 50 ng/mL BMP4 100 µg/mL 40 ng/mL VEGF 100 µg/mL 50 ng/mL 1. Prepare mesoderm induction medium 2 fresh and allow it to warm to room temperature. 2. Remove the plate containing spheroids from the incubator. 3. Gently swirl the plate so all spheroids cluster to the middle of the well. 4. Tilt the plate at an ~45° angle for 4–5 minutes to ensure spheroids settle via gravity sedimentation. 5. With the plate tilted, carefully remove 1 mL of culture medium from each well by using a P1000 pipette. Note: For medium replacement in a vertical wheel bioreactor (100 mL working volume), remove vessel from the platform for 5–10 minutes to ensure spheroids settle via gravity sedimentation. Manually aspirate 50 mL of medium. Add an equal volume (50 mL) of fresh mesoderm induction medium 2 to replace the aspirated volume. 6. Replace the 1 mL spent medium with equal volume prewarmed mesoderm induction medium 2 per well. 7. Incubate the plate at 37°C, 5% CO₂ on an orbital shaker platform set to 70 RPM or a vertical wheel bioreactor set to 40 RPM. Note: iHPC induction can be initiated between days 1–3. Due to differences in PSC line nucleation efficiency and proliferation, spheroid growth may vary. We recommend initiating iHPC induction when most PSC spheroids are between 150–250 µm in diameter. A B 4 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Day 2: iHPC induction iHPC induction medium Component Stock conc. Final conc. StemPro-34 basal medium 1X 1X StemPro-34 supplement 40X 1X 1-thioglycerol 11.5 M 400 µmol/L L-(+)-Ascorbic Acid 10 mg/mL 20 µg/mL CTS GlutaMAX-I Supplement 200 mM 2 mM ITS 100X 1X SCF 100 µg/mL 50 ng/mL BMP4 100 µg/mL 40 ng/mL VEGF 100 µg/mL 50 ng/mL bFGF 100 µg/mL 10 ng/mL 1. Prepare iHPC induction medium fresh (without growth factors or cytokines) and filter before use. The medium is stable for 30 days when stored in the dark at 4–8°C. Growth factors and cytokines are added immediately prior to medium changes. Note: Working volumes vary by vessel type (Table 4). Table 4. Volume of iHPC induction medium 1, based on vessel type. Working volume for different vessels Vessel Non–tissue culture treated 6-well plate Non–tissue culture treated 125 mL flask Non–tissue culture treated 100 mL bioreactor Volume 3 mL 20 mL 100 mL 2. Gently swirl the plate so all the spheroids cluster to the middle of the well. 3. Tilt the plate at an ~45° angle for 4–5 minutes to ensure spheroids settle via gravity sedimentation. 4. With the plate still tilted, carefully remove ~80% of the medium from each well by using a P1000 pipette. Note: For medium replacement in a vertical wheel bioreactor, remove the vessel from the platform for 5–10 minutes to allow spheroids to settle via gravity sedimentation. Manually aspirate 50 mL of media. Add an equal volume (i.e. 50 mL) of iHPC induction medium to replace the aspirated volume. 5. Add enough iHPC induction medium to obtain the appropriate working volume, as listed in step 1. 6. Incubate the plate at 37°C, 5% CO₂ on an orbital shaker platform set to 60 RPM (or 30–40 RPM for vertical wheel bioreactor cultures). 5 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Day 5: iHPC full medium change iHPC maturation medium 1 Component Stock conc. Final conc. StemPro-34 basal medium 1X 1X StemPro-34 supplement 40X 1X 1-thioglycerol 11.5 M 400 µmol/L L-(+)-Ascorbic Acid 10 mg/mL 20 µg/mL CTS GlutaMAX-I Supplement 200 mM 2 mM ITS 100X 1X SCF 100 µg/ml 50 ng/mL FLT3 100 µg/mL 20 ng/mL TPO 100 µg/mL 30 ng/mL 1. Prepare iHPC maturation medium 1 fresh and allow it to warm to room temperature. 2. Gently swirl the plate so all the spheroids cluster to the middle of the well. 3. Tilt the plate at an ~45° angle for 4–5 minutes to ensure spheroids settle via gravity sedimentation. 4. Manually remove as much medium per well as possible using a 1,000 µL or 10 mL pipette. Be careful not to remove any spheroids. Notes: • For medium replacement in a vertical wheel bioreactor, remove vessel from the platform for 5–10 minutes to ensure spheroids settle via gravity sedimentation. Manually aspirate 50 mL of medium. Add an equal volume (50 mL) of iHPC maturation medium 1 to replace the aspirated volume. • If spheroids are accidentally aspirated, add the spheroid suspension back to the well and repeat steps 1–3 to minimize sample loss. 5. Add 3 mL iHPC maturation medium 1 to each well. 6. Incubate the plate at 37°C, 5% CO₂ on an orbital shaker platform set to 60 RPM (or 30–40 RPM for bioreactor cultures). 6 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Day 8: iHPC half medium change iHPC maturation medium 2 Component Stock conc. Final conc. StemPro-34 basal medium 1X 1X StemPro-34 supplement 40X 1X 1-thioglycerol 11.5 M 400 µmol/L L-(+)-Ascorbic Acid 10 mg/mL 20 µg/mL CTS GlutaMAX-I Supplement 200 mM 2 mM ITS 100X 1X SCF 100 µg/mL 100 ng/mL FLT3 100 µg/mL 40 ng/mL TPO 100 µg/mL 60 ng/mL 1. Prepare iHPC maturation medium 2 fresh and allow it to warm to room temperature. 2. Gently swirl the plate so all the spheroids cluster to the middle of the well. 3. Tilt the plate at an ~45° angle for 4–5 minutes to ensure spheroids settle via gravity sedimentation. 4. Medium change is needed while minimizing any loss of maturing iHPCs. With the plate tilted, carefully remove 1.5 mL of culture medium from each well and transfer to a 15 mL conical tube. Be careful not to disturb the settled spheroids. Note: For medium replacement in a vertical wheel bioreactor, remove vessel from the platform for 5 minutes to ensure spheroids settle via gravity sedimentation. Remove 50 mL medium without disturbing the settled spheroids and put in a 50 mL tube. 5. Centrifuge the conical tube at 300 x g for 5 minutes at room temperature. 6. Aspirate the supernatant without disturbing the cell pellet. 7. Resuspend the cell pellet in 1.5 mL of iHPC maturation medium 2. 8. Add cell suspension back into the same well. 9. Incubate the plate at 37°C, 5% CO2 on an orbital shaker platform set to 60 RPM (or 30–40 RPM for bioreactor cultures). Notes: Assessing iHPC phenotype: • To assess phenotype, we recommend culturing duplicate samples: one sample for iHPC phenotyping, and one sample to continue to iNK cell differentiation. • When optimizing this protocol, we recommend phenotyping for hematopoietic surface markers between days 6 and 8. Once a baseline has been established, this step can be optimized. • To the sample used for iHPC phenotyping, gently break apart cell clumps by triturating with a P1000 pipette ~10 times. There will be some clumps remaining that will get discarded in the next step. • Pass the entire 3 mL from one well through a 40 µm cell strainer into a conical tube. • Wash the well with 2 mL DMEM/F-12 and filter through the same 40 µm cell strainer. • Wash the cell strainer with 15 mL DMEM/F-12 to ensure all single cells have passed through. • Proceed with a flow cytometry protocol using the harvested single cell suspension, assessing for the following markers: CD34, CD45, CD90, CD43. 7 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Day 9: Initiate iNK cell differentiation Schedule for iNK cell differentiation: Day 9 Day 12 Day 15–38 Full medium change Half medium change Half medium change every 2–3 days Check iNK cell phenotype once a week beginning on day 25 iNK cell induction: iNK cell induction medium Component Stock conc. Final conc. CTS NK-Xpander Basal Medium 1X 56% MEMα 1X 28% Human AB Serum 1X 15% CTS NK-Xpander Supplement 50X 1X β-mercaptoethanol 11.5 M 1 µM CTS GlutaMAX-I Supplement 200 mM 4 mM IL-3 100 µg/mL 5 ng/mL SCF 100 µg/mL 20 ng/mL FLT3 100 µg/mL 10 ng/mL IL-7 100 µg/mL 20 ng/mL IL-15 100 µg/mL 10 ng/mL 1. Prepare iNK cell induction medium fresh (without growth factors or cytokines) and filter before use. Medium is stable for 30 days when stored in the dark at 4–8°C. Growth factors and cytokines are added immediately prior to medium changes. 2. Calculate the volume of iNK cell induction medium required (Table 5). Table 5. Seeding volume based on vessel type. PSC to iHPC vessel Recommended iHPC to iNK cell vessel Seeding volume 6-well plate Non–tissue culture treated 6-well plate 3 mL/well 125 mL flask Non–tissue culture treated T-75 flask 15 mL 100 mL vertical wheel bioreactor Non–tissue culture treated T-175 flask 50 mL 3. Collect the entire volume of iNK cell induction medium (including residual spheroids) from each well. Place into separate conical tubes for each well. 4. To the emptied culture vessel, immediately add 2 mL of iNK cell induction medium to each well. 5. Centrifuge the conical tubes containing cells and supernatant at 300 x g for 5 minutes at room temperature. 6. Aspirate supernatant. 7. Resuspend the cell pellet in 1 mL of iNK cell induction medium. 8. Transfer resuspended cells back into the original well. Notes: 125 mL flask: While the entire volume of the vessel is being centrifuged, add 10 mL of iNK induction medium to a non–tissue culture treated T-75 flask. Aspirate the supernatant and resuspend the cell pellet in 5 mL of iNK cell induction medium. Transfer the resuspended cells back into the new T-75 flask. 100 mL bioreactor: While the entire volume of the vessel is being centrifuged, add 40 mL of iNK induction medium to a non–tissue culture treated T-175 flask. Aspirate the supernatant and resuspend the cell pellet in 10 mL of iNK cell induction medium. Transfer the resuspended cells back into the new T-175 flask. 9. Incubate plates at 37°C, 5% CO2 for static culture. Figure 3. Morphological observations during iNK differentiation. (A) Day 12: Individual iHPC and spheroids begin attaching to vessel floor. (B) Day 21: Spheroids form a “feeder-like” layer to which the suspension iNK cells adhere and further mature. (C) Day 30: Further expansion of the spheroid monolayer and expansion of iNK cells. The iNK cells at this point will be loosely attached and may reside in suspension. A B C 8 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Day 12: iNK cell differentiation half medium change iNK cell induction medium Component Stock conc. Final conc. CTS NK-Xpander Basal Medium 1X 56% MEM α 1X 28% Human AB Serum 1X 15% CTS NK-Xpander Supplement 50X 1X β-mercaptoethanol 11.5 M 1 µM CTS GlutaMAX-I Supplement 200 mM 4 mM IL-3 100 µg/mL 10 ng/mL SCF 100 µg/mL 40 ng/mL FLT3 100 µg/mL 20 ng/mL IL-7 100 µg/mL 40 ng/mL IL-15 100 µg/mL 20 ng/mL 1. Prepare iNK cell induction medium fresh (without growth factors or cytokines) and filter before use. Medium is stable for 30 days when stored in the dark at 4–8°C. Growth factors and cytokines are added immediately prior to medium changes. 2. Collect 1.5 mL of culture medium from each well and transfer to a 15 mL conical tube. 3. Centrifuge the tubes at 300 x g for 5 minutes at room temperature. 4. Aspirate supernatant. 5. Resuspend cells in 1.5 mL of iNK cell induction medium per well to replace the aspirated volume. 6. Transfer resuspended cells back into the original well/vessel. Notes: T-75 flask: Collect 8 mL of culture medium and transfer to a 15 mL conical tube. Centrifuge the tubes at 300 x g for 5 minutes, then aspirate supernatant. Resuspend in 8 mL of iNK cell induction medium to replace the aspirated volume. Transfer resuspended cells back into the original vessel. T-175 flask: Collect 25 mL of culture medium and transfer to a 25 mL conical tube. Centrifuge the tubes at 300 x g for 5 minutes then aspirate supernatant. Resuspend in 25 mL of iNK cell induction medium to replace the aspirated volume. Transfer resuspended cells back into the original vessel. 7. Incubate plates/flasks at 37°C, 5% CO₂ for static culture. 9 CTS StemScale PSC Suspension Medium thermofisher.com/ctsstemscale Day 15: iNK cell differentiation half medium change iNK cell maturation medium Component Stock conc. Final conc. CTS NK-Xpander Basal Medium 1X 56% MEM α 1x 28% Human AB Serum 1x 15% CTS NK-Xpander Supplement 50X 1X β-mercaptoethanol 11.5 M 1 µM CTS GlutaMAX-I Supplement 200 mM 4 mM SCF 100 µg/mL 40 ng/mL FLT3 100 µg/mL 20 ng/mL IL-7 100 µg/mL 40 ng/mL IL-15 100 µg/mL 20 ng/mL 1. Prepare iNK cell maturation medium fresh (without growth factors or cytokines) and filter before use. Medium is stable for 30 days when stored in the dark at 4–8°C. Growth factors and cytokines are added immediately prior to medium changes. 2. Calculate the volume of iNK cell maturation medium required for a half medium change. 3. Collect 1.5 mL of culture medium from each well and transfer to a 15 mL conical tube. 4. Centrifuge the tubes at 300 x g for 5 minutes at room temperature. 5. Aspirate supernatant. 6. Resuspend cells in 1.5 mL of iNK cell maturation medium per well to replace the aspirated volume. 7. Transfer resuspended cells back into the original well/vessel. Notes: T-75 flask: Collect 8 mL of culture medium and transfer to a 15 mL conical tube. Spin tubes at 300 x g for 5 minutes then aspirate supernatant. Resuspend in 8 mL of iNK cell maturation medium to replace the aspirated volume. Transfer resuspended cells back into the original vessel. T-175 flask: Collect 25 mL of culture medium and transfer to 25 mL conical tube. Spin tubes at 300 x g for 5 minutes then aspirate supernatant. Resuspend in 25 mL of iNK cell differentiation medium to replace the aspirated volume. Transfer resuspended cells back into the original vessel. 8. Incubate plates/flasks at 37°C, 5% CO2 for static culture. Day 18–36: iNK cell differentiation half medium change 1. Calculate the volume of iNK cell maturation medium required for a half medium change. 2. Collect 1.5 mL of culture medium from each well and transfer to a 15 mL conical tube. 3. Centrifuge the tubes at 300 x g for 5 minutes at room temperature. 4. Aspirate supernatant. 5. Resuspend cells in 1.5 mL of iNK cell maturation medium per well to replace the aspirated volume. 6. Transfer resuspended cells back into the original well. Notes: T-75 flask: Collect 8 mL of culture medium and transfer to a 15 mL conical tube. Spin tubes at 300 x g for 5 minutes then aspirate supernatant. Resuspend in 8 mL of iNK cell maturation medium to replace the aspirated volume. Transfer resuspended cells back into the original vessel. T-175 flask: Collect 25 mL of culture medium and transfer to 25 mL conical tube. Spin tubes at 300 x g for 5 minutes then aspirate supernatant. Resuspend in 25 mL of iNK cell differentiation medium to replace the aspirated volume. Transfer resuspended cells back into the original vessel. 7. Incubate plates/flasks at 37°C, 5% CO2 for static culture. 8. Perform medium changes every 2–3 days. Notes: We recommend performing flow cytometry weekly starting the week of day 25, checking for CD45 and CD56 expression to monitor differentiation. Collect aliquots of the cell suspension from the well plate/v