Extracellular Vesicle Collection from Human Stem Cells Grown in Suspension Bioreactors

Abstract

Extracellular vesicles (EVs) are particles with 100–1000 nm sizes which are secreted by cells for intercellular communication. Meanwhile, studies have found that EVs secreted by human stem cells carry similar characteristics (microRNAs, proteins, metabolites, etc.) from their cell counterpart. Thus, EVs derived from stem cells, especially human induced pluripotent stem cells (hiPSCs) and human mesenchymal stromal/stem cells (hMSCs) are promising candidates for cell-free therapy. However, conventional planar culture is insufficient to produce a large amount of cells or EVs to satisfy clinical requirements. In this chapter, we described feasible approaches to harvest EVs secreted by lineage-specific hiPSCs and undifferentiated hMSCs in suspension bioreactors. Differentiation of hiPSCs to cortical organoids can be performed in suspension bioreactors and the corresponding EVs can be isolated and purified. This scale-up protocol can be applied to a majority of stem cell types with EV collection thus provides useful information for both experimental and biomanufacturing purposes.

1. Introduction

In past decades, human stem cells including pluripotent stem cells and adult multipotent stem cells have drawn significant attentions pre-clinically and clinically [1]. Human induced pluripotent stem cells (hiPSCs) exhibit robust differentiation potential for modeling of disease pathology, drug discovery, and act as potential cell sources for therapeutic applications [2]. On the other hand, human mesenchymal stromal/stem cells (hMSCs) have been widely acknowledged for their regenerative potentials mediated by their secretome and paracrine effects. Studies have demonstrated promising strategies by using both types of stem cells to understand disease progression and therapeutic mechanisms, as well as in preclinical/clinical trials following biomanufacturing regulations [3, 4]. Recently, extracellular vesicles (EVs) or exosomes, the nano-particles secreted by cells, have been shown to carry specific cellular characteristics from the original cells and thus could represent lineage commitment or exhibit potential therapeutic effects [5]. EVs generated from undifferentiated hiPSCs carry specific cargos indicating the difference from the original cells being reprogrammed [6]. Potentially, EVs from hiPSCs exert homeostatic regulation on stressed human umbilical vein endothelial cells (HUVECs) to maintain cell viability and reduce senescence [7]. Moreover, EVs from hiPSC-derived neural progenitor cells (hiPSC-NPCs) not only carry lineage-specific information but also exhibit pro-neurogenesis and circuit assembly potentials [8, 9]. For hMSCs, there is an increasing body of evidence indicating the secreted EVs capture the therapeutic potentials of hMSCs and remain biologically active after transplantation [10–12]. Human umbilical cord MSC-secreted EVs have been shown to exert liver protection under culture stress and ameliorate autoimmune symptoms via immunomodulation in rodent uveoretinitis models [13, 14]. Clinical trials have approached to study steroid refractory graft-versus host disease and grade III-IV chronic kidney disease patients with hMSC-EV administrations [15, 16].

Two-dimension (2D) conventional culture of human stem cells is sufficient and robust in general laboratory scale. However, industrial or clinical applications require a large amount of as well as stem cell derivatives beyond what the 2D culture can provide. For instance, 2–8 × 10 cells/kg patient weight would be needed for graft-versus-host diseases in stem cell based therapy [17]. To fulfill the clinical requirement, three-dimensional (3D) suspension bioreactors are designed for biomanufacturing of human stem cells and their derivatives. Microcarriers have been applied for anchorage-dependent cells such as hMSCs. Studies have reported up to 43-fold increase of hMSCs in a 50 L stir-tank bioreactor [18]. Moreover, advanced bioreactors, such as wave bioreactor and PBS vertical wheel (PBS-VW) bioreactors have been designed to reduce shear stress to maintain hMSC homeostasis while still provide rapid expansion [19]. With the success of cell expansion in bioreactors, stem cell derivatives such as EVs should also be able to be scaled up for biomanufacturing purpose, though not many cases have been published.

In this protocol chapter, detailed procedures and handling notes are described for EV isolation from hiPSC-NPCs and human umbilical cord-derived MSCs (hUC-MSCs) grown in suspension bioreactors. For hiPSC-NPC differentiation and expansion, a 50 mL spinner flask is selected. For hUC-MSC expansion, Cytodex-1 microcarrier and a 100 mL PBS-VW bioreactor is selected. EV-free medium preparation and the downstream EV isolation is modified and described based on our previous studies [20–22].

2. Materials

2.1. Materials for hiPSC Differentiation in Planar and Bioreactor Cultures

1. A frozen hiPSC line, iPSK3 cells, was kindly provided by Dr. Stephen Duncan from Medical College of Wisconsin [23]. Briefly, the cell line was derived from human foreskin fibroblasts with transfection of plasmid DNA encoding reprogramming factors: OCT4, NANOG, SOX2, and LIN28 (see Note 1).

2. Prior to hiPSC culture, Lactose Dehydrogenase Elevating Virus (LDEV)-Free Reduced Growth Factor Basement Membrane Matrix Geltrex (Life Technologies, #A1413202) is thawed under 4 °C overnight, then aliquoted and stored under −20 °C. Geltrex is diluted in cold Dulbecco’s Modified Eagle’s medium (DMEM, Life Technologies, Carlsbad, CA) at 1:100 dilution (1%) and stored under −20 °C. Tissue culture surface is coated with 1% Geltrex solution for overnight in 37 °C incubator (see Note 2).

3. hiPSC complete culture medium (hiPSC-CCM) is prepared by adding 20% serum-free mTeSR™1 5× supplement (StemCell™ Technologies Inc., #85852) in mTeSR™1 basal medium (StemCell™ Technologies Inc., #05850). The hiPSC-CCM can be stored under 4 °C. To dissociate hiPSCs for passaging, Accutase solution (VWR International, Radnor, PA., #10210-214) is used for obtaining single cell suspension. Aliquoted Accutase solution is stored under −20 °C. Rho-associated kinase (ROCK) inhibitor Y27632, 2 mg or 10 mM in 624.4 μL dimethyl sulfoxide (DMSO, #MD-0025), is purchased from iXCells Biotechnologies (San Diego, CA). The final working concentration is 10 μM by adding 1 μL of the stock solution per mL of hiPSC-CCM (see Note 3).

4. Human NPC differentiation medium is composed of DMEM-F12 (Gibco™, #125000-062), with 2% B27 serum-free supplement (50×, Life Technologies, #17504044) and stored under 4 °C. Dual-SMAD inhibition for neural differentiation of hiPSCs is achieved by two small molecules: SB431542 (Sigma, #S4317) and LDN193189 (Sigma, #SML0559), both dissolved in DMSO and stored under −20 °C. The working concentrations in differentiation medium are 10 μM SB431542 and 100 nM LDN193189 (see Note 4).

2.2. Materials for hMSC Expansion in Planar and Bioreactor Cultures

1. Frozen hMSC lines derived from human umbilical cords are provided by SynergyBiologics (Tallahassee, FL). hMSCs at passage 0 (P0) are cryopreserved and stored in liquid nitrogen.

2. hMSC complete culture medium (hMSC-CCM) is prepared by dissolving 10.08 g Minimum Essential Medium Alpha Medium (Life Technologies, #12000-063), 2.2 g sodium bicarbonate (Sigma-Aldrich, #S5761), 10 mL penicillin-streptomycin (Life Technologies, #15070-063), and 100 mL fetal bovine serum (FBS, Atlanta Biologicals, Inc. #S11110) in 1 L deionized water; then filtered by 0.2 μm pore size filter in sterile bottle and stored under 4 °C for future use. The 150 mm diameter tissue culture petri dishes are obtained from VWR International (#25382-442). 0.25% trypsin/EDTA solution is from ThermoFisher Scientific (#25200056).

2.3. Bioreactor Preparation

1. A lab scale of 50 mL glass spinner flask bioreactor (Wheaton, #356875) is used for hiPSC-NPC differentiation, as well as medium collection for EV isolation. Prior to culture, the glass vessel is coated with 1 mL sigmacote (Sigma-Aldrich, #SL2) and then dried overnight at room temperature. The spinner flask is autoclaved for future use.

2. For PBS-VW bioreactor, PBS mini system is purchased from PBS Biotech™, Inc. (Camarillo, CA). This system includes magnetic agitation base and a sterile single-use vessel in 100 mL. The system is assembled and placed in standard cell culture 37 °C incubator with 5% CO₂. Cytodex 1 microcarriers (GE Healthcare Life Sciences, #17-0448-01) are prepared by hydrating the microcarriers in phosphate buffered saline (PBS) for overnight and washed twice with PBS before autoclave. Then the microcarriers are washed again with PBS and ready to use.

2.4. Materials for EV Isolation from Human Stem Cells Grown in Bioreactor Cultures

1. EV-free FBS is prepared by ultracentrifuge. FBS is spun at 4 °C, 100,000 × g for 20 h. The supernatant is carefully collected as EV-free FBS for EV collection.

2. All centrifuges are pre-cooled to 4 °C during EV isolation and purification. Polyethylene glycol 6000 (PEG 6000, VWR International, Radnor, PA., #80503) solution is prepared by mixing 160 g PEG6000 with 1 M sodium chloride (NaCl, VWR International, #470302) in 1 L milli-Q water, then filtered with 0.2 μm pore size filter, resulting in 16% PEG6000 solution. EV-free PBS is prepared by double-filter of sterile PBS for future use (see Note 5).

3. Methods

3.1. Culture and Expansion of hiPSCs in Planar Culture

1. Frozen iPSK3 cells are recovered by immediately thawing in a 37 °C water bath for 30 s until a small piece of ice remains. Spray the cryopreserve vial with 70% ethanol and open the vial in biological safety cabinet. Transfer the cell suspension carefully into at least ten times of volume of hiPSC-CCM (for instance, 1 mL cell suspension in 10 mL CCM) in a centrifuge tube. Gently pipette the mixture and then centrifuge at 300 × g for 5 min (see Note 6).

2. After centrifugation, carefully remove the supernatant and do not disturb the cell pellet. Resuspend the cell pellet with 1–3 mL hiPSC-CCM carefully and distribute the cell suspension onto Geltrex-coated culture surface at 1–2 × 10⁵ cells/cm². ROCK inhibitor Y27632 is added at 10 μM in the media. The recovered hiPSCs are cultured in a standard incubator (37 °C, 5% CO₂).

3. First medium change is performed 24-h later to remove ROCK inhibitor. Cell attachment can be visualized under microscope. hiPSC culture is maintained in CCM and medium is replaced every 2–4 days. When cells are compacted and reach a high density, medium change is performed daily.

4. For passaging hiPSCs, culture medium is removed, and the cells are washed with sterile PBS. Then the cells are incubated with Accutase solution at 37 °C for 5 min. Gently pipet the mixture to acquire single cell suspension. Transfer the mixture to a 15 mL centrifuge tube, wash the culture surface once with hiPSC-CCM, and transfer the medium to the centrifuge tube (see Note 7).

5. Spin down the cells at 300 × g for 5 min and remove the supernatant. Resuspend the cell pellet with hiPSC-CCM and determine the cell number by hemocytometer. Passaged hiPSCs can be expanded on Geltrex-coated surface or prepared for NPC differentiation in bioreactors.

3.2. Differentiation of NPC Organoid from hiPSCs in Spinner Flasks

1. hiPSC suspension is collected and seeded in the 15 mL spinner flask at 4–5 × 10⁵ cells/mL in NPC differentiation medium containing 10 μM Y27632. The bioreactor is set up on a programmable magnetic stirrer (Wheaton, #900701) and the whole system is placed in a standard culture incubator (37 °C, 5% CO₂).

2. In initial aggregation phase (day 0), intermittent agitation is used after cell seeding in the bioreactor. The stirrer is set to 80 rpm for 15 min and off for 15 min for a total of 10 cycles. Then the agitation speed is set to 80 rpm for the rest of the culture.

3. At day 1, stop the agitation and let the hiPSC aggregates to settle down at the bottom; carefully remove the medium by pipette and resuspend the aggregates with fresh NPC differentiation medium containing 10 μM SB431542 and 100 nM LDN193189 to induce neural lineage commitment. Restart the agitation and culture.

4. Medium sample can be collected every day for the analysis of glucose and lactate concentration by YSI2950 biochemical analyzer. Half medium change is performed every 2 days with fresh NPC differentiation medium containing 10 μM SB431542 and 100 nM LDN193189.

5. On day 8, NPC aggregates or spheres can be collected for the evaluation of neural progenitor markers. Both SB431542 and LDN193189 are removed by medium change and fresh NPC differentiation medium is added to the bioreactor for continuous agitation culture (see Note 8).

6. For EV collection, conditioned medium collection can be performed by defined time interval, for example, every 2 days. Cultures can be maintained in this manner for a very long time (25–70 days) (Fig. 1a). Collected media are preserved under 4 °C for further processing.

Fig. 1.

(a) hiPSC-NPC organoid growth in spinner flask at day 25 and day 27 under optical light microscope. Scale bar: 400 μm. (b) Transmission electron microscopy (TEM) images of EVs from hiPSC-NPC bioreactor culture. Scale bar: 200 nm. (c) The size of hiPSC-NPC secreted EVs determined by nanoparticle tracking analysis (NTA)

3.3. Expansion of hMSCs in Planar Culture

1. One vial of frozen hMSCs (generally contains 1 × 10⁶ cells) is recovered by immediately thawing in a 37 °C water bath for 30 s until a small piece of ice remains. Spray the vial with 70% ethanol and open the vial in biological safety cabinet. Transfer the cell suspension carefully into at least ten times of volume of hMSC-CCM (for instance, 1 mL cell suspension in 10 mL CCM) in centrifuge tube. Gently pipette the mixture and then centrifuge at 400 × g for 5 min.

2. After centrifugation, carefully remove the supernatant and do not disturb the cell pellet. Resuspend the cell pellet with 1–3 mL hMSC-CCM carefully and distribute the cell suspension into 150 mm diameter petri dish at 1200–1500 cells/cm². The culture is maintained in a standard incubator (37 °C, 5% CO₂).

3. First medium change is performed after 12 h to remove debris and unattached cells. Then medium is changed every 2 days with fresh hMSC-CCM.

4. For passaging hMSCs, culture medium is removed, and the cells are washed with sterile PBS twice. Then the hMSCs are treated with 0.25% trypsin solution at 37 °C for 5–7 min. Trypsin is neutralized by adding hMSC-CCM and detached cells are collected in a 15 mL centrifuge tube. The petri dish is washed with fresh hMSC-CCM once to collect residue cells for maximized yield.

5. Spin down the hMSCs at 500 × g for 5 min and remove the supernatant. Resuspend the cell pellet with hMSC-CCM and determine the cell number by hemocytometer. hMSCs can be expanded on tissue culture surface or on microcarriers in PBS-VW bioreactors for EV collection (see Note 9).

3.4. Expansion of hMSCs in PBS-VW Bioreactors

1. hMSCs in planar cultures are harvested and resuspended with hMSC-CCM containing EV-free FBS. Then hMSCs are mixed with sterile Cytodex-1 microcarriers (0.25–0.5 g) at the density of 1000–1500 cells/cm². The mixture is added to the PBS-VW bioreactors and the volume is brought to 60 mL with hMSC-CCM containing EV-free FBS (see Note 10).

2. On day 0, intermittent agitation is used after cells and microcarriers are transferred into the bioreactor. The agitation base is set to 25 rpm for 5 min and off for 15 min for a total of 12 cycles (roughly 4 h). Then the agitation speed is set to 25 rpm for the rest of the culture period. The total volume is remained at 60 mL for seeding phase. After seeding, the total volume is brought to 100 mL with fresh hMSC-CCM containing EV-free FBS. The culture is maintained in a standard incubator (37 °C, 5% CO₂).

3. Sampling of the bioreactor can be performed every day by taking 1 mL of mixture from PBS-VW bioreactor under agitation. Cells on microcarriers can be visualized by nucleus staining (Fig. 2a) and culture medium supernatants can be used for glucose/lactate measurement.

4. Medium collection is performed every 2 days with 50% fresh medium change. The agitation is stopped to let cells/microcarriers to settle down. 50 mL media are collected from the PBS-VW bioreactor. Then 50 mL of pre-warmed EV-free hMSC-CCM is added to the bioreactor and agitation is started again. Collected medium is ready for EV isolation.

Fig. 2.

(a) hUC-MSCs growth in PBS-VW bioreactors at day 1 and day 7, stained by Hoechst 33342 for cell nuclei and visualized under fluorescent microscope. Scale bar: 200 μm. (b) Transmission electron microscopy (TEM) images of EVs from hUC-MSC bioreactor culture. Scale bar: 100 nm. (c) The size of hUC-MSC secreted EVs determined by nanoparticle tracking analysis (NTA)

3.5. EV Isolation from the Collected Media

1. Collected media can be stored at 4 °C for 1 week before processing, otherwise they should be stored at −50 °C. Collected media, both from hiPSC-NPC organoid culture and hMSC bioreactor culture, undergo modified differential centrifugation at 4 °C: 500 × g for 5 min, 2000 × g for 10 min, and 10,000 × g for 30 min. Supernatants are collected from each step (see Note 11).

2. For hiPSC-NPC organoid conditioned media, 10% EV-free FBS and 16% PEG6000 solution are added to the medium to a final concentration of 8% PEG6000. For hMSC conditioned media, only 16% PEG6000 solution is added to the media at a final concentration of 8% PEG6000. All samples are well mixed and stored at 4 °C for 12–24 h.

3. Next, medium samples are centrifuged at 3200 × g for 60 min at 4 °C to collect PEG-enriched EV pellets. The supernatants are discarded, and the pellets are resuspended in sterile, EV-free PBS for washing out PEG 6000, then ultracentrifuge is performed at 120,000 × g for 70 min at 4 °C.

4. After ultracentrifuge, supernatants are discarded and the EV pellets are resuspended in sterile, EV-free PBS (usually at 100 μL, but the volume can vary). The morphology of the isolated EVs can be examined by transmission electron microscopy (Figs. 1b and 2b). EV size distribution and concentrations can be determined by Nanoparticle Tracking Analysis (NTA) using a Malvern NanoSight LM10 instrument (Figs. 1c and 2c) (see Note 12).

5. Stem cell-secreted EVs are aliquoted and stored at −80 °C for further downstream analysis, culture experiments, or animal transplantation.